Methods for selectively stimulating proliferation of T cells

ABSTRACT

Methods for inducing a population of T cells to proliferate by activating the population of T cells and stimulating an accessory molecule on the surface of the T cells with a ligand which binds the accessory molecule are described. T cell proliferation occurs in the absence of exogenous growth factors or accessory cells. T cell activation is accomplished by stimulating the T cell receptor (TCR)/CD3 complex or the CD2 surface protein. To induce proliferation of an activated population T cells, an accessory molecule on the surface of the T cells, such as CD28, is stimulated with a ligand which binds the accessory molecule. The T cell population expanded by the method of the invention can be genetically transduced and used for immunotherapy or can be used in methods of diagnosis.

RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.08/403,253, filed Mar. 10, 1995, entitled “Methods for SelectivelyStimulating Proliferation of T Cells” (now U.S. Pat. No. 6,352,694);which in turn is a continuation-in-part of U.S. Ser. No. 08/253,964,filed Jun. 3, 1994, entitled “Methods for Selectively StimulatingProliferation of T Cells” (currently pending). The contents of theaforementioned applications are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

The development of techniques for propagating T cell populations invitro has been crucial to many of the recent advances in theunderstanding of T cell recognition of antigen and T cell activation.The development of culture methods for the generation of humanantigen-specific T cell clones has been useful in defining antigensexpressed by pathogens and tumors that are recognized by T cells toestablish methods of immunotherapy to treat a variety of human diseases.Antigen-specific T cells can be expanded in vitro for use in adoptivecellular immunotherapy in which infusions of such T cells have beenshown to have anti-tumor reactivity in a tumor-bearing host. Adoptiveimmunotherapy has also been used to treat viral infections inimmunocompromised individuals.

Techniques for expanding human T cells in vitro have relied on the useof accessory cells and exogenous growth factors, such as IL-2. The useof IL-2 and, for example, an anti-CD3 antibody to stimulate T cellproliferation is known to expand the CD8⁺ subpopulation of T cells. Therequirement for MHC-matched antigen presenting cells as accessory cellspresents a significant problem for long-term culture systems. Antigenpresenting cells are relatively short lived. Thus, in a long-termculture system, antigen presenting cells must be continuously obtainedfrom a source and replenished. The necessity for a renewable supply ofaccessory cells is problematic for treatment of immunodeficiencies inwhich accessory cells are affected. In addition, when treating viralinfection, accessory cells which may carry the virus may result incontamination of the entire T cell population during long term culture.An alternative culture method to clone and expand human T cells in vitroin the absence of exogenous growth factor and accessory cells would beof significant benefit.

SUMMARY OF THE INVENTION

This invention pertains to methods for selectively inducing ex vivoexpansion of a population of T cells in the absence of exogenous growthfactors, such as lymphokines, and accessory cells. In addition, T cellproliferation can be induced without the need for antigen, thusproviding an expanded T cell population which is polyclonal with respectto antigen reactivity. The method provides for sustained proliferationof a selected population of CD4⁺ or CD8⁺ T cells over an extended periodof time to yield a multi-fold increase in the number of these cellsrelative to the original T cell population.

According to the method of the invention, a population of T cells isinduced to proliferate by activating the T cells and stimulating anaccessory molecule on the surface of the T cells with a ligand whichbinds the accessory molecule. Activation of a population of T cells isaccomplished by contacting the T cells with a first agent whichstimulates a TCR/CD3 complex-associated signal in the T cells.Stimulation of the TCR/CD3 complex-associated signal in a T cell isaccomplished either by ligation of the T cell receptor (TCR)/CD3 complexor the CD2 surface protein, or by directly stimulating receptor-coupledsignaling pathways. Thus, an anti-CD3 antibody, an anti-CD2 antibody, ora protein kinase C activator in conjunction with a calcium ionophore isused to activate a population of T cells.

To induce proliferation, an activated population of T cells is contactedwith a second agent which stimulates an accessory molecule on thesurface of the T cells. For example, a population of CD4⁺ T cells can bestimulated to proliferate with an anti-CD28 antibody directed to theCD28 molecule on the surface of the T cells. Alternatively, CD4⁺ T cellscan be stimulated with a natural ligand for CD28, such as B7-1 and B7-2.The natural ligand can be soluble, on a cell membrane, or coupled to asolid phase surface. Proliferation of a population of CD8⁺ T cells isaccomplished by use of a monoclonal antibody ES5.2D8 which binds to CD9,an accessory molecule having a molecular weight of about 27 kD presenton activated T cells. Alternatively, proliferation of an activatedpopulation of T cells can be induced by stimulation of one or moreintracellular signals which result from ligation of an accessorymolecule, such as CD28.

The agent providing the primary activation signal and the agentproviding the costimulatory agent can be added either in soluble form orcoupled to a solid phase surface. In a preferred embodiment, the twoagents are coupled to the same solid phase surface.

Following activation and stimulation of an accessory molecule on thesurface of the T cells, the progress of proliferation of the T cells inresponse to continuing exposure to the ligand or other agent which actsintracellularly to simulate a pathway mediated by the accessory moleculeis monitored. When the rate of T cell proliferation decreases, the Tcells are reactivated and restimulated, such as with additional anti-CD3antibody and a co-stimulatory ligand, to induce further proliferation.In one embodiment, the rate of T cell proliferation is monitored byexamining cell size. Alternatively, T cell proliferation is monitored byassaying for expression of cell surface molecules in response toexposure to the ligand or other agent, such as B7-1 or B7-2. Themonitoring and restimulation of the T cells can be repeated forsustained proliferation to produce a population of T cells increased innumber from about 100- to about 100,000-fold over the original T cellpopulation.

The method of the invention can be used to expand selected T cellpopulations for use in treating an infectious disease or cancer. Theresulting T cell population can be genetically transduced and used forimmunotherapy or can be used for in vitro analysis of infectious agentssuch as HIV. Proliferation of a population of CD4⁺ cells obtained froman individual infected with HIV can be achieved and the cells renderedresistant to HIV infection. Following expansion of the T cell populationto sufficient numbers, the expanded T cells are restored to theindividual. Similarly, a population of tumor-infiltrating lymphocytescan be obtained from an individual afflicted with cancer and the T cellsstimulated to proliferate to sufficient numbers and restored to theindividual. In addition, supernatants from cultures of T cells expandedin accordance with the method of the invention are a rich source ofcytokines and can be used to sustain T cells in vivo or ex vivo.

The invention also pertains to compositions comprising an agent thatprovides a costimulatory signal to a T cell for T cell expansion (e.g.,an anti-CD28 antibody, B7-1 or B7-2 ligand), coupled to a solid phasesurface which may additionally include an agent that provides a primaryactivation signal to the T cell (e.g., an anti-CD3 antibody) coupled tothe same solid phase surface. These agents are preferably attached tobeads. Compositions comprising each agent coupled to different solidphase surfaces (i.e., an agent that provides a primary T cell activationsignal coupled to a first solid phase surface and an agent that providesa costimulatory signal coupled to a second solid phase surface) are alsowithin the scope of this invention. Furthermore, the invention provideskits comprising the compositions, including instructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts in vitro growth curves of CD4⁺ peripheral blood T cellsin response to culture with either an anti-CD3 antibody andinterleukin-2 (IL-2) (●-●), an anti-CD3 antibody and an anti-CD28antibody mAb 9.3 (⋄-⋄) or PHA only (Δ-Δ).

FIG. 2 depicts the growth curve of CD4⁺ peripheral blood T cellscultured in fetal calf serum and either anti-CD3 antibodies and IL-2 ( -) or an anti-CD3 antibody and an anti-CD28 antibody, mAb 9.3 (⋄-⋄).

FIG. 3 depicts the growth curves of CD4⁺ peripheral blood T cellscultured in the presence of phorbol myristic acid (PMA) and ionomycinwith or without IL-2, or with an anti-CD28 antibody, mAb 9.3. Thesymbols are as follows: PMA and ionomycin (P⁺I) is represented by (□);PMA, ionomycin and IL-2 (P⁺I⁺IL-2) is represented by (●); and PMA,ionomycin and anti-CD28 antibody (P⁺I⁺9.3) is represented by (♦).

FIG. 4 is a schematic representation of the selective expansion of CD4⁺T cells following CD28 stimulation in comparision to T cell stimulationwith IL-2.

FIG. 5 depicts fluorescent activated cell sorter analysis (FACS) inwhich cells were stained after isolation (day 0, panel A), or after 26days in culture with either CD28 stimulation (panel B) or IL-2 culture(panel C), with phycoerythrin conjugated anti-CD3, CD4, CD8 or with anIgG2a control monoclonal antibody and fluorescence quantified with aflow cytometer.

FIG. 6 shows FACS analysis of the EX5.3D10 monoclonal antibody depictingreactivity with CD28 in comparison to an anti-CD28 monoclonal antibody9.3. The following cell lines were tested: Panel A, untransfectedCHO-DG44 cells; Panel B, CHO—HH cells; Panel C, unactivated peripheralblood lymphocytes; and Panel D, Jurkat No. 7 cells.

FIG. 7 shows FACS analysis of the ES5.2D8 monoclonal antibody depictingthe binding reactivity with the following cell lines: Panel A, CHO-DG44cells; Panel B, CHO-105A cells; Panel C, unactivated human peripheralblood lymphocytes; and Panel D, PMA activated peripheral bloodlymphocytes.

FIG. 8 is a photograph depicting immunoprecipitation analysis ofdetergent lysates of surface labeled human activated T cells indicatingthat monoclonal antibody ES5.2D8 reacts with a 27 kD cell surfaceprotein.

FIG. 9 depicts the increases in mean cell volume of CD4⁺ T cellsfollowing stimulation (S1, S2, S3, S4, S5 and S6) with an anti-CD3monoclonal antibody and an anti-CD28 monoclonal antibody over days inculture.

FIG. 10 depicts the cyclic expression of B7-1 on CD4⁺ T cells followingstimulation (S1, S2, S3, S4, S5 and S6) with an anti-CD3 monoclonalantibody and an anti-CD28 monoclonal antibody over days in culture.

FIG. 11 is a bar graph depicting the amount of IL-2 produced by CD4⁺ Tcells following stimulation with an anti-CD3 monoclonal antibody and ananti-CD28 monoclonal antibody or IL-2 over days in culture.

FIG. 12 is a bar graph depicting the amount of granulocyte-macrophagecolony-stimulating factor (GM-CSF) produced by CD4⁺ T cells followingstimulation with an anti-CD3 monoclonal antibody and an anti-CD28monoclonal antibody or IL-2 over days in culture.

FIG. 13 is a bar graph depicting the amount of tumor necrosis factor(TNF) produced by CD4⁺ T cells following stimulation with an anti-CD3monoclonal antibody and an anti-CD28 monoclonal antibody or IL-2 overdays in culture.

FIG. 14 is a bar graph depicting the T cell receptor (TCR) diversity inCD4⁺ T cells following stimulation with an anti-CD3 monoclonal antibodyand an anti-CD28 monoclonal antibody at day 1 and day 24 of culture.

FIG. 15 depicts cell surface staining of CD4⁺ T cells obtained from anHIV seronegative individual following stimulation (S1, S2 and S3) withan anti-CD3 monoclonal antibody and an anti-CD28 monoclonal antibodyover days in culture.

FIG. 16 depicts cell surface staining of CD4⁺ T cells obtained from anHIV seropositive individual following stimulation (S1, S2 and S3) withan anti-CD3 monoclonal antibody and an anti-CD28 monoclonal antibodyover days in culture.

FIG. 17 depicts expansion of CD8⁺ T cells following stimulation with ananti-CD3 monoclonal antibody and an monoclonal antibody ES5.2D8 at day 4and day 7 of culture.

FIG. 18 depicts FACS analysis with the monoclonal antibody ES5.2D8(panels C and D) or a control IgG (panels A and B) depicting the bindingreactivity with MOP cells transfected with a plasmid encoding the CD9antigen.

FIG. 19 depicts CD28⁺ T cell expansion following stimulation withanti-CD3 monoclonal antibody coated beads and anti-CD28 antibody, B7-1,B7-2, B7-1 and B7-2, or control CHO-neo cells at different time pointsafter stimulation.

FIG. 20 depicts CD28⁺ T cell expansion following stimulation with PMAand anti-CD28 antibody, B7-1, B7-2, B7-1 and B7-2, or control CHO-neocells at different time points after stimulation.

FIG. 21 depicts CD28⁺ T cell expansion following stimulation withanti-CD3 monoclonal antibody coated beads, B7-1, B7-2, B7-1 and B7-2, orcontrol CHO-neo cells in the presence of various amounts of antiCD28 Fabfragments and the amount of IL-2 secreted in the medium.

FIG. 22 depicts growth curves of CD4⁺ peripheral blood T cells in longterm cultures with either anti-CD3 monoclonal antibody, anti-CD28antibody, B7-1, B7-2, or control CHO-neo cells.

FIG. 23 shows the amounts of IL-2 and IL-4 produced from CD4⁺ T cells(panels A and B), CD4⁺/CD45RA⁺ T cells (panels C and D) and CD4⁺/CD45RO⁺T cells (panels E and F) stimulated with anti-CD3 monoclonal antibodycoated beads and B7-1, B7-2, or control CHO-neo cells at the indicatedCHO cell to T cell ratio.

FIG. 24 shows the amounts of interferon-gamma and IL-4 produced fromCD4⁺ T cells (panels A and B), CD4⁺/CD45RA⁺ T cells (panels C and D) andCD4⁺/CD45RO⁺ T cells (panels E and F) stimulated with anti-CD3monoclonal antibody coated beads and B7-1, B7-2, or control CHO-neocells at the indicated CHO cell to T cell ratio.

FIG. 25 shows the amounts of IL-2 (panel A) and IL-4 (panel B) producedfrom CD4⁺ T cells stimulated with medium alone, anti-CD3 monoclonalantibody and B7-1, B7-2, control CHO-neo cells after the first round ofstimulation (day 1) or a second round of stimulation (day 12).

FIG. 26 shows amounts of interferon-gamma (panel A) and IL-5 (panel B)produced from CD4⁺ T cells stimulated with medium alone, anti-CD3monoclonal antibody and B7-1, B7-2, control CHO-neo cells after thefirst round of stimulation (day 1) or a second round of stimulation (day12).

FIG. 27 shows amounts of TNF-alpha (panel A) and GM-CSF (panel B)produced from CD4⁺ T cells stimulated with medium alone, anti-CD3monoclonal antibody and B7-1, B7-2, control CHO-neo cells after thefirst round of stimulation (day 1) or a second round of stimulation (day12).

FIG. 28 shows the amounts of IL-2 (panel A) and IL-4 (panel B) secretedfrom CD28⁺ T cells stimulated with medium alone, anti-CD3 and anti-CD28antibody coated beads (“cis”), anti-CD3 and anti-CD28 antibody coatedbeads and control CHO-neo cells (“cis”⁺CHO-neo), anti-CD3 coated beadsand B7-1 CHO cells (anti-CD3⁺CHO-B7-1), or anti-CD3 coated beads andanti-CD28 coated beads (anti-CD3⁺CD28 “trans”) after initial stimulation(day 1), second stimulation (day 2), or third stimulation (day 3).

FIG. 29 depicts growth curves of the CD28⁺ T cells stimulated in theexperiment shown in FIG. 28.

FIG. 30 depicts growth curves of CD4⁺ T cells from an HIV infectedindividual stimulated with anti-CD3 and anti-CD28 antibodies in thepresence (A/D/N) or absence (No Drug) of anti-retroviral drugs.

FIG. 31 depicts growth curves of CD4⁺ T cells stimulated with anti-CD3and anti-CD28 coated beads in the presence of added IL-2 (OKT3⁺IL-2) orin the absence of added IL-2 (OKT3⁺9.3) in large scale cultures.

DETAILED DESCRIPTION OF THE INVENTION

The methods of this invention enable the selective stimulation of a Tcell population to proliferate and expand to significant numbers invitro in the absence of exogenous growth factors or accessory cells.Interaction between the T cell receptor (TCR)/CD3 complex and antigenpresented in conjunction with either major histocompatibility complex(MHC) class I or class II molecules on an antigen-presenting cellinitiates a series of biochemical events termed antigen-specific T cellactivation. The term “T cell activation” is used herein to define astate in which a T cell response has been initiated or activated by aprimary signal, such as through the TCR/CD3 complex, but not necessarilydue to interaction with a protein antigen. A T cell is activated if ithas received a primary signaling event which initiates an immuneresponse by the T cell.

T cell activation can be accomplished by stimulating the T cell TCR/CD3complex or via stimulation of the CD2 surface protein. An anti-CD3monoclonal antibody can be used to activate a population of T cells viathe TCR/CD3 complex. Although a number of anti-human CD3 monoclonalantibodies are commercially available, OKT3 prepared from hybridomacells obtained from the American Type Culture Collection or monoclonalantibody G19-4 is preferred. Similarly, binding of an anti-CD2 antibodywill activate T cells. Stimulatory forms of anti-CD2 antibodies areknown and available. Stimulation through CD2 with anti-CD2 antibodies istypically accomplished using a combination of at least two differentanti-CD2 antibodies. Stimulatory combinations of anti-CD2 antibodieswhich have been described include the following: the T11.3 antibody incombination with the T11.1 or T11.2 antibody (Meuer, S. C. et al. (1984)Cell 36:897-906) and the 9.6 antibody (which recognizes the same epitopeas T11.1) in combination with the 9-1 antibody (Yang, S. Y. et al.(1986) J. Immunol. 137:1097-1100). Other antibodies which bind to thesame epitopes as any of the above described antibodies can also be used.Additional antibodies, or combinations of antibodies, can be preparedand identified by standard techniques.

A primary activation signal can also be delivered to a T cell throughuse of a combination of a protein kinase C (PKC) activator such as aphorbol ester (e.g., phorbol myristate acetate) and a calcium ionophore(e.g., ionomycin which raises cytoplasmic calcium concentrations). Theuse of these agents bypasses the TCR/CD3 complex but delivers astimulatory signal to T cells. These agents are also known to exert asynergistic effect on T cells to promote T cell activation and can beused in the absence of antigen to deliver a primary activation signal toT cells.

Although stimulation of the TCR/CD3 complex or CD2 molecule is requiredfor delivery of a primary activation signal in a T cell, a number ofmolecules on the surface of T cells, termed accessory or costimulatorymolecules have been implicated in regulating the transition of a restingT cell to blast transformation, and subsequent proliferation anddifferentiation. Thus, in addition to the primary activation signalprovided through the TCR/CD3 complex, induction of T cell responsesrequires a second, costimulatory signal. One such costimulatory oraccessory molecule, CD28, is believed to initiate or regulate a signaltransduction pathway that is distinct from those stimulated by the TCRcomplex.

Accordingly, to induce an activated population of T cells to proliferate(i.e., a population of T cells that has received a primary activationsignal) in the absence of exogenous growth factors or accessory cells,an accessory molecule on the surface of the T cell, such as CD28, isstimulated with a ligand which binds the accessory molecule or with anagent which acts intracellularly to stimulate a signal in the T cellmediated by binding of the accessory molecule. In one embodiment,stimulation of the accessory molecule CD28 is accomplished by contactingan activated population of T cells with a ligand which binds CD28.Activation of the T cells with, for example, an anti-CD3 antibody andstimulation of the CD28 accessory molecule results in selectiveproliferation of CD4⁺ T cells. An anti-CD28 monoclonal antibody orfragment thereof capable of crosslinking the CD28 molecule, or a naturalligand for CD28 (e.g., a member of the B7 family of proteins, such asB7-1(CD80) and B7-2 (CD86) (Freedman, A. S. et al. (1987) J. Immunol.137:3260-3267; Freeman, G. J. et al. (1989) J. Immunol. 143:2714-2722;Freeman, G. J. et al. (1991) J. Exp. Med. 174:625-63 1; Freeman, G. J.et al. (1993) Science 262:909-911; Azuma, M. et al. (1993) Nature366:76-79; Freeman, G. J. et al. (1993) J. Exp. Med. 178:2185-2192)) canbe used to induce stimulation of the CD28 molecule. In addition, bindinghomologues of a natural ligand, whether native or synthesized bychemical or recombinant technique, can also be used in accordance withthe invention. Ligands useful for stimulating an accessory molecule canbe used in soluble form, attached to the surface of a cell, orimmobilized on a solid phase surface as described herein. Anti-CD28antibodies of fragments thereof useful in stimulating proliferation ofCD4⁺ T cells include monoclonal antibody 9.3, an IgG2a antibody (Dr.Jeffery Ledbetter, Bristol Myers Squibb Corporation, Seattle, Wash.),monoclonal antibody KOLT-2, an IgG1 antibody, 15E8, an IgG1 antibody,248.23.2, an IgM antibody and EX5.3D10, an IgG2a antibody. In onespecific embodiment, the molecule providing the primary activationsignal, for example a molecule which provides stimulation through theTCR/CD3 complex or CD2, and the costimulatory molecule are coupled tothe same solid phase support. In particular, T cell activation andcostimulation can be provided by a solid phase surface containinganti-CD3 and anti-CD28 antibodies.

A preferred anti-CD28 antibody is monoclonal antibody 9.3 or EX5.3D10.The EX5.3D10 monoclonal antibody was derived from immunizing a Balb/cmouse with CHO (Chinese hamster ovary) cells transfected with the humanCD28 gene (designated CHO-hh). Hybridomas from the fusion were selectedby whole cell ELISA screening against Jurkat (human T leukemia) CD28tranfectants designated Jurkat #7. Reactivity of the EX5.3D10 with CD28was further confirmed by fluorescent activated cell sorter analysis(FACS) analysis in which it was tested side by side with the monoclonal9.3 (FIG. 6). Neither antibody bound to untransfected CHO-DG44 cells andtheir binding profiles were nearly identical for the two CD28transfectant lines, CHO-hh and Jurkat #7, as well as normal humanperipheral blood lymphocytes. A hybridoma which produces the monoclonalantibody EX5.3D10 has been deposited with the American Type CultureCollection on Jun. 4, 1993, at ATCC Deposit No. HB11373.

In a specific embodiment of the invention, activated T cells arecontacted with a stimulatory form of a natural ligand for CD28 forcostimulation. The natural ligands of CD28 include the members of the B7family of proteins, such as B7-1 (CD80) (SEQ ID NO:1 and 2) and B7-2(CD86) (SEQ ID NO:3 and 4). B7-1 and B7-2 are collectively referred toherein as “B7 molecules”. A “stimulatory form of a natural ligand forCD28” is a form of a natural ligand that is able to bind to CD28 andcostimulate the T cell. Costimulation can be evidenced by proliferationand/or cytokine production by T cells that have received a primaryactivation signal, such as stimulation through the CD3/TCR complex orthrough CD2.

Expression or Coupling of B7 Molecules on the Surface of Cells

In a preferred embodiment of the invention, a B7 molecule is localizedon the surface of a cell. This can be accomplished by transfecting acell with a nucleic acid encoding the B7 molecule (e.g. B7-1, B7-2) in aform suitable for its expression on the cell surface or alternatively bycoupling a B7 molecule to the cell surface.

The B7 molecules are preferably expressed on the surface of a cell bytransfection of the cell with a nucleic acid encoding the B7 molecule ina form suitable for expression of the molecule on the surface of thecell. The terms “transfection” or “transfected with” refers to theintroduction of exogenous nucleic acid into a mammalian cell andencompass a variety of techniques useful for introduction of nucleicacids into mammalian cells including electroporation, calcium-phosphateprecipitation, DEAE-dextran treatment, lipofection, microinjection andinfection with viral vectors. Suitable methods for transfectingmammalian cells can be found in Sambrook et al. (Molecular Cloning: ALaboratory Manual, 2 nd Edition, Cold Spring Harbor Laboratory press(1989)) and other laboratory textbooks. The nucleic acid to beintroduced may be, for example, DNA encompassing the gene(s) encodingB7-1 and/or B7-2, sense strand RNA encoding B7-1 and/or B7-2 or arecombinant expression vector containing a cDNA encoding B7-1 and/orB7-2. The nucleotide sequence of a cDNA encoding human B7-1 is shown inSEQ ID NO: 1, and the amino acid sequence of a human B7-1 protein isshown in SEQ ID NO:2. The nucleotide sequence of a cDNA encoding humanB7-2 is shown in SEQ ID NO: 3, and the amino acid sequence of a humanB7-2 protein is shown in SEQ ID NO:4. The nucleic acids encoding B7-1and B7-2 are further described in Freedman, A. S. et al. (1987) J.Immunol. 137:3260-3267; Freeman, G. J. et al. (1989) J. Immunol.143:2714-2722; Freeman, G. J. et al. (1991) J. Exp. Med. 14:625-631;Freeman, G. J. et al. (1993) Science 262:909-911; Azuma, M. et al.(1993) Nature 366:76-79 and; Freeman, G. J. et al. (1993) J. Exp. Med.178:2185-2192.

The nucleic acid is in a form suitable for expression of the B7 moleculein which the nucleic acid contains all of the coding and regulatorysequences required for transcription and translation of a gene, whichmay include promoters, enhancers and polyadenylation signals, andsequences necessary for transport of the molecule to the surface of thetumor cell, including N-terminal signal sequences. When the nucleic acidis a cDNA in a recombinant expression vector, the regulatory functionsresponsible for transcription and/or translation of the cDNA are oftenprovided by viral sequences. Examples of commonly used viral promotersinclude those derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40, and retroviral LTRs. Regulatory sequences linked to thecDNA can be selected to provide constitutive or inducible transcription,by, for example, use of an inducible promoter, such as themetallothionin promoter or a glucocorticoid-responsive promoter.Expression of B7-1 or B7-2 on the surface of a cell can be accomplished,for example, by including the native transmembrane coding sequence ofthe molecule in the nucleic acid sequence, or by including signals whichlead to modification of the protein, such as a C-terminalinositol-phosphate linkage, that allows for association of the moleculewith the outer surface of the cell membrane.

The B7 molecule can be expressed on a cell using a plasmid expressionvector which contains nucleic acid, e.g., a cDNA, encoding the B7molecule. Suitable plasmid expression vectors include CDM8 (Seed, B.,Nature 329, 840 (1987)) and pMT2PC (Kaufman, et al., EMBO J. 6, 187-195(1987)). Since only a small fraction of cells (about 1 out of 10⁵)typically integrate transfected plasmid DNA into their genomes, it isadvantageous to transfect a nucleic acid encoding a selectable markerinto the tumor cell along with the nucleic acid(s) of interest.Preferred selectable markers include those which confer resistance todrugs such as G418, hygromycin and methotrexate. Selectable markers maybe introduced on the same plasmid as the gene(s) of interest or may beintroduced on a separate plasmid. Following selection of transfectedcells using the appropriate selectable marker(s), expression of thecostimulatory molecule on the surface of the cell can be confirmed byimmunofluorescent staining of the cells. For example, cells may bestained with a fluorescently labeled monoclonal antibody reactiveagainst the costimulatory molecule or with a fluorescently labeledsoluble receptor which binds the costimulatory molecule such as CTLA41g.Expression of the B7 costimulatory molecule can be determined using amonoclonal antibody, such as BB1 or 133, which recognizes B7-1 or themonoclonal antibody IT2 which recognizes B7-2. Alternatively, a labeledsoluble CD28 or CTLA4 protein or fusion protein (e.g., CTLA41g) whichbinds to the B7 molecules can be used to detect expression of B7 on thecell surface.

The cell to be transfected can be any eukaryotic cell, preferably cellsthat allow high level expression of the transfected gene, such aschinese hamster ovary (CHO) cells or COS cells. The cell is mostpreferably a CHO cell and a specific protocol for transfection of thesecells is provided in Example 11.

In another embodiment, B7 molecules (e.g., B7-1, B7-2) are coupled tothe cell surface by any of a variety of different methods. In thisembodiment, the B7 molecule to be coupled to the cell surface can beobtained using standard recombinant DNA technology and expressionsystems which allow for production and isolation of the costimulatorymolecule(s) or obtained from a cell expressing the costimulatorymolecule, as described below for the preparation of a soluble form ofthe B7 molecules. The isolated costimulatory molecule is then coupled tothe cell. The terms “coupled” or “coupling” refer to a chemical,enzymatic or other means (e.g., antibody) by which the B7 molecule islinked to a cell such that the costimulatory molecule is present on thesurface of the cell and is capable of triggering a costimulatory signalin T cells. For example, the B7 molecule can be chemically crosslinkedto the cell surface using commercially available crosslinking reagents(Pierce, Rockford Ill.). Another approach to coupling a B7 molecule to acell is to use a bispecific antibody which binds both the costimulatorymolecule and a cell-surface molecule on the cell. Fragments, mutants orvariants of a B7 molecule which retain the ability to trigger acostimulatory signal in T cells when coupled to the surface of a cellcan also be used.

The level of B7 molecules expressed on or coupled to the cell surfacecan be determined by FACS analysis, as described in Example 11.

For T cell costimulation, the B7-expressing cells can be cultured to ahigh density, mitomycin C treated (e.g., at 25 μg/ml for an hour),extensively washed, and incubated with the T cells to be costimulated.The ratio of T cells to B7-expressing cells can be anywhere between 10:1to 1:1, preferably 2.5:1 T cells to B7-expressing cells.

Soluble forms of B7 molecules as costimulator

The natural ligands of CD28 can also be presented to T cells in asoluble form. Soluble forms of B7 molecules include natural B7 molecules(e.g., B7-1, B7-2), a fragment thereof, or modified form of the fulllength or fragment of the B7 molecule that is able to bind to CD28 andcostimulate the T cell. Costimulation can be evidenced by proliferationand/or cyotkine production by T cells that have received a primaryactivation signal. Modifications of B7 molecules include modificationsthat preferably enhance the affinity of binding of B7 molecules to CD28molecules, but also modifications that diminish or do not affect theaffinity of binding of B7 molecules to CD28 molecules. Modifications ofB7 molecules also include those that increase the stability of a solubleform of a B7 molecule. The modifications of B7 molecules are usuallyproduced by amino acid substitutions, but can also be produced bylinkage to another molecule.

In one specific embodiment, the soluble form of a B7 molecule is afusion protein containing a first peptide consisting of a B7 molecule(e.g., B7-1, B7-2), or fragment thereof and a second peptidecorresponding to a moiety that alters the solubility, binding, affinity,stability, or valency (i.e., the number of binding sites available permolecule) of the first peptide. Preferably, the first peptide includesan extracellular domain portion of a B7 molecule (e.g., about amino acidresidues 24-245 of the B7-2 molecule having an amino acid sequence shownin SEQ ID NO: 4) that interacts with CD28 and is able to provide acostimulatory signal as evidenced by stimulation of proliferation of Tcells or secretion of cytokines from the T cells upon exposure to theB71g fusion protein and a primary T cell activation signal. Thus, aB7-lIg fusion protein will comprise at least about amino acids 1-208(SEQ ID NO:2) of B7-1 and a B7-2Ig fusion protein will comprise at leastabout amino acids 24-245 (SEQ ID NO:4) of B7-2.

The second peptide is a fragment of an Ig molecule, such as an Fcfragment that comprises the hinge, CH2 and CH3 regions of human IgG1 orIgG4. Several Ig fusion proteins have been previously described (seee.g., Capon, D. J. et al. (1989) Nature 337:525-531 and Capon U.S. Pat.No. 5,116,964 [CD4-IgG1 constructs]; Linsley, P. S. et al. (1991) J.Exp. Med. 173:721-730 [a CD28-IgG1 construct and a B7-1-IgG1 construct];and Linsley, P. S. et al. (1991) J. Exp. Med. 174:561-569 [a CTLA4-IgG1]). A resulting B71g fusion protein (e.g., B7-1Ig, B7-2Ig) may havealtered B7-2solubility, binding affinity, stability, or valency and mayincrease the efficiency of protein purification. In particular fusion ofa B7 molecule or portion thereof to the Fc region of an immunoglobulinmolecule generally provides an increased stability to the protein, inparticular in the plasma.

Fusion proteins within the scope of the invention can be prepared byexpression of a nucleic acid encoding the fusion protein in a variety ofdifferent systems. Typically, the nucleic acid encoding a B7 fusionprotein comprises a first nucleotide sequence encoding a first peptideconsisting of a B7 molecule or a fragment thereof and a secondnucleotide sequence encoding a second peptide corresponding to a moietythat alters the solubility, binding, stability, or valency of the firstpeptide, such as an immunoglobulin constant region. Nucleic acidencoding a peptide comprising an immunoglobulin constant region can beobtained from human immunoglobulin mRNA present in B lymphocytes. It isalso possible to obtain nucleic acid encoding an immunoglobulin constantregion from B cell genomic DNA. For example, DNA encoding Cλ1 or Cλ4 canbe cloned from either a cDNA or a genomic library or by polymerase chainreaction (PCR) amplification in accordance standard protocols. Apreferred nucleic acid encoding an immunoglobulin constant regioncomprises all or a portion of the following: the DNA encoding human Cλ1(Takahashi, N. S. et al. (1982) Cell 29:671-679), the DNA encoding humanCλ2; the DNA encoding human Cλ3 (Huck, S., et al. (1986) Nucl. Acid Res.14: 1779); and the DNA encoding human Cλ4. When an immunoglobulinconstant region is used in the B7 fusion protein, the constant regioncan be modified to reduce at least one constant region mediatedbiological effector function. For example, DNA encoding a Cλ1 or Cλ4constant region can be modified by PCR mutagenesis or site directedmutagenesis. Protocols and reagents for site directed mutagenesissystems can be obtained commercially from Amersham International PLC,Amersham, UK.

In a particularly prefered embodiment of the invention, B7-1Ig andB7-2Ig fusion proteins comprise about amino acids 1-208 of B7-1 (SEQ IDNO: 2) and about amino acids 24-245 of B7-2 (SEQ ID NO: 4),respectively, fused to the heavy chain of IgG1.

In one embodiment the first and second nucleotide sequences are linked(i.e., in a 5′ to 3′ orientation by phosphodiester bonds) such that thetranslational frame of the B7 protein or fragment thereof and the IgC(i.e., Fc fragment that comprises the hinge, CH2, and CH3 regions ofhuman IgG) coding segments are maintained (i.e., the nucleotidesequences are joined together in-frame). Thus, expression (i.e.,transcription and translation) of the nucleotide sequence produces afunctional B7Ig fusion protein. The nucleic acids of the invention canbe prepared by standard recombinant DNA techniques. For example, a B7Igfusion protein can be constructed using separate template DNAs encodingB7 and an immunoglobulin constant region. The appropriate segments ofeach template DNA can be amplified by polymerase chain reaction (PCR)and ligated in frame using standard techniques. A nucleic acid of theinvention can also be chemically synthesized using standard techniques.Various methods of chemically synthesizing polydeoxynucleotides areknown, including solid-phase synthesis which has been automated incommercially available DNA synthesizers (See e.g., Itakura et al. U.S.Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No. 4,458,066; andItakura U.S. Pat. Nos. 4,401,796 and 4,373,071, incorporated byreference herein).

The nucleic acids encoding B7 molecules or B7Ig fusion proteins (e.g.,B7-1, B7-2) can be inserted into various expression vectors, which inturn direct the synthesis of the corresponding protein in a variety ofhosts, particularly eucaryotic cells, such as mammalian or insect cellculture and procaryotic cells, such as E. coli. Expression vectorswithin the scope of the invention comprise a nucleic acid as describedherein and a promotor operably linked to the nucleic acid. Suchexpression vectors can be used to transfect host cells to therebyproduce fusion proteins encoded by nucleic acids as described herein. Anexpression vector of the invention, as described herein, typicallyincludes nucleotide sequences encoding a B7 molecule or B7Ig fusionprotein operably linked to at least one regulatory sequence. “Operablylinked” is intended to mean that the nucleotide sequence is linked to aregulatory sequence in a manner which allows expression of thenucleotide sequence in a host cell (or by a cell extract). Regulatorysequences are art-recognized and can be selected to direct expression ofthe desired protein in an appropriate host cell. The term regulatorysequence is intended to include promoters, enhancers, polyadenylationsignals and other expression control elements. Such regulatory sequencesare known to those skilled in the art and are described in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). It should be understood that the design of theexpression vector may depend on such factors as the choice of the hostcell to be transfected and/or the type and/or amount of protein desiredto be expressed.

An expression vector of the invention can be used to transfect cells,either procaryotic or eucaryotic (e.g., mammalian, insect or yeastcells) to thereby produce fusion proteins encoded by nucleotidesequences of the vector. Expression in procaryotes is most often carriedout in E. coli with vectors containing constitutive or induciblepromotors. Certain E. coli expression vectors (so called fusion-vectors)are designed to add a number of amino acid residues to the expressedrecombinant protein, usually to the amino terminus of the expressedprotein. Such fusion vectors typically serve three purposes: 1) toincrease expression of recombinant protein; 2) to increase thesolubility of the target recombinant protein; and 3) to aid in thepurification of the target recombinant protein by acting as a ligand inaffinity purification. Examples of fusion expression vectors includepGEX (Amrad Corp., Melbourne, Australia) and pMAL (New England Biolabs,Beverly, Mass.) which fuse glutathione S-tranferase and maltose Ebinding protein, respectively, to the target recombinant protein.Accordingly, a B7 molecule or B7Ig fusion gene may be linked toadditional coding sequences in a procaryotic fusion vector to aid in theexpression, solubility or purification of the fusion protein. Often, infusion expression vectors, a proteolytic cleavage site is introduced atthe junction of the fusion moiety and the target recombinant protein toenable separation of the target recombinant protein from the fusionmoiety subsequent to purification of the fusion protein. Such enzymes,and their cognate recognition sequences, include Factor Xa, thrombin andenterokinase.

Inducible non-fusion expression vectors include pTrc (Amann et al.,(1988) Gene 69:301-315) and pET 11d (Studier et al., Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990) 60-89). Target gene expression from the pTrc vector4 relies onhost RNA polymerase transcription from the hybrid trp-lac fusionpromoter. Target gene expression from the pET 11d vector relies ontranscription from the T7 gn10-lac 0 fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21 (DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gn1 under the transcriptional control of thelacUV 5 promoter.

One strategy to maximize expression of at B7 molecule or B7Ig fusionprotein in E. coli is to express the protein in a host bacteria with animpaired capacity to proteolytically cleave the recombinant protein(Gottesman, S., Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990) 119-128). Another strategywould be to alter the nucleotide sequence of the B7 molecule or B7Igfusion protein construct to be inserted into an expression vector sothat the individual codons for each amino acid would be thosepreferentially utilized in highly expressed E. coli proteins (Wada etal., (1992) Nuc. Acids Res. 20:2111-2118). Such alteration of nucleicacid sequences are encompassed by the invention and can be carried outby standard DNA synthesis techniques.

Alternatively, a B7 molecule or B7Ig fusion protein can be expressed ina eucaryotic host cell, such as mammalian cells (e.g., Chinese hamsterovary cells (CHO) or NS0 cells), insect cells (e.g., using a baculovirusvector) or yeast cells. Other suitable host cells may be found inGoeddel, (1990) supra or are known to those skilled in the art.Eucaryotic, rather than procaryotic, expression of a B7 molecule or B7Igmay be preferable since expression of eucaryotic proteins in eucaryoticcells can lead to partial or complete glycosylation and/or formation ofrelevant inter- or intra-chain disulfide bonds of a recombinant protein.For expression in mammalian cells, the expression vector's controlfunctions are often provided by viral material. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40. To express a B7 molecule or B7Ig fusion protein inmammalian cells, generally COS cells (Gluzman, Y., (1981) Cell23:175-182) are used in conjunction with such vectors as pCDM8 (Seed,B., (1987) Nature 329:840) for transient amplification/expression, whileCHO (dhfr Chinese Hamster Ovary) cells are used with vectors such aspMT2PC (Kaufman et al. (1987), EMBO J. 6:187-195) for stableamplification/expression in mammalian cells. A preferred cell line forproduction of recombinant protein is the NS0 myeloma cell line availablefrom the ECACC (catalog #85110503) and described in Galfre, G. andMilstein, C. ((1981) Methods in Enzymology 73(13):3-46; and Preparationof Monoclonal Antibodies: Strategies and Procedures, Academic Press,N.Y., N.Y). Examples of vectors suitable for expression of recombinantproteins in yeast (e.g., S. cerivisae) include pYepSec1 (Baldari. etal., (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYES2(Invitrogen Corporation, San Diego, Calif.). Baculovirus vectorsavailable for expression of proteins in cultured insect cells (SF 9cells) include the pAc series (Smith et al., (1983) Mol. Cell Biol.3:2156-2165) and the pVL series (Lucklow, V. A., and Summers, M. D.,(1989) Virology 170:31-39).

Vector DNA can be introduced into procaryotic or eucaryotic cells viaconventional transformation or transfection techniques such as calciumphosphate or calcium choloride co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, or electroporation. Suitable methods fortransforming host cells can be found in Sambrook et al. (MolecularCloning: A Laboratory Manual, 2 nd Edition, Cold Spring HarborLaboratory press (1989)), and other laboratory textbooks.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfaction of cells may integrate DNA into their genomes. In order toidentify and select these integrants, a gene that encodes a selectablemarker (e.g., resistance to antibiotics) is generally introduced intothe host cells along with the gene of interest. Preferred selectablemarkers include those which confer resistance to drugs, such as G418,hygromycin and methotrexate. Nucleic acid encoding a selectable markermay be introduced into a host cell on the same plasmid as the gene ofinterest or may be introduced on a separate plasmid. Cells containingthe gene of interest can be identified by drug selection (e.g., cellsthat have incorporated the selectable marker gene will survive, whilethe other cells die). The surviving cells can then be screened forproduction of B7 molecules or B7Ig fusion proteins by, for example,immunoprecipitation from cell supernatant with an anti-B7 monoclonalantibody.

B7 molecules or B7 Ig fusion proteins produced by recombinant techniquemay be secreted and isolated from a mixture of cells and mediumcontaining the protein. Alternatively, the protein may be retainedcytoplasmically and the cells harvested, lysed and the protein isolated.A cell culture typically includes host cells, media and otherbyproducts. Suitable mediums for cell culture are well known in the art.Protein can be isolated from cell culture medium, host cells, or bothusing techniques known in the art for purifying proteins.

For T cell costimulation, the soluble forms of the natural ligands forCD28 are added to the T cell culture in an amount sufficient to resultin costimulation of activated T cells. The appropriate amount of solubleligand to be added will vary with the specific ligand, but can bedetermined by assaying different amounts of the soluble ligand in T cellcultures and measuring the extent of costimulation by proliferationassays or production of cytokines, as described in the Examples.

Coupling of the Natural Ligands to a Solid Phase Surface

In another embodiment of the invention, a natural ligand of CD28 (B7-1,B7-2) can be presented to T cells in a form attached to a solid phasesurface, such as beads. The B7 molecules, fragments thereof or modifiedforms thereof capable of binding to CD28 and costimulating the T cells(e.g., B7 fusion proteins) can be prepared as described for the solubleB7 forms. These molecules can then be attached to the solid phasesurface via several methods. For example the B7 molecules can becrosslinked to the beads via covalent modification using tosyl linkage.In this method, B7 molecules or B7 fusion proteins are in 0.05M boratebuffer, pH 9.5 and added to tosyl activated magnetic immunobeads (DynalInc., Great Neck, N.Y.) according to manufacturer's instructions. Aftera 24 hr incubation at 22° C., the beads are collected and washedextensively. It is not mandatory that immunmagnetic beads be used, asother methods are also satisfactory. For example, the B7 molecules mayalso be immobilized on polystyrene beads or culture vessel surfaces.Covalent binding of the B7 molecules or B7Ig fusion proteins to thesolid phase surface is preferrable to adsorption or capture by asecondary monoclonal antibody. B7Ig fusion proteins can be attached tothe solid phase surface through anti-human IgG molecules bound to thesolid phase surface. In particular, beads to which anti-human IgGmolecules are bound can be obtained from Advanced Magnetics, Inc. Thesebeads can then be incubated with the B7Ig fusion proteins in anappropriate buffer such as PBS for about an hour at 5° C., and theuncoupled B7Ig proteins removed by washing the beads in a buffer, suchas PBS.

It is also possible to attach the B7 molecules to the solid phasesurface through an avidin- or streptavidin-biotin complex. In thisparticular embodiment, the soluble B7 molecule is first crosslinked tobiotin and then reacted with the solid phase surface to which avidin orstreptavidin molecules are bound. It is also possible to crosslink theB7 molecules with avidin or streptavidin and to react these with a solidphase surface that is covered with biotin molecules.

The amount of B7 molecules attached to the solid phase surface can bedetermined by FACS analysis if the solid phase surface is that of beadsor by ELISA if the solid phase surface is that of a tissue culture dish.Antibodies reactive with the B7 molecules, such as mAb BBI, mAb IT2, andmAb 133 can be used in these assays.

In a specific embodiment, the stimulatory form of a B7 molecule isattached to the same solid phase surface as the agent that stimulatesthe TCR/CD3 complex, such as an anti-CD3 antibody. In addition toanti-CD3, other antibodies that bind to receptors that mimic antigensignals may be used, for example, the beads or other solid phase surfacemay be coated with combinations of anti-CD2 and a B7 molecule.

In a typical experiment, B7-coated beads or beads coated with B7molecules and an agent that stimulates the TCR/CD3 complex will be addedat a ratio of 3 beads per T cell.

Agents Which Act Intracellularly to Stimulate a Signal Associated withCD28 Ligation

In another embodiment of the invention, an activated population of CD4⁺T cells is stimulated to proliferate by contacting the T cells with anagent which acts intracellularly to stimulate a signal in the T cellmediated by ligation of an accessory molecule, such as CD28. The term“agent”, as used herein, is intended to encompass chemicals and otherpharmaceutical compounds which stimulate a costimulatory or other signalin a T cell without the requirement for an interaction between a T cellsurface receptor and a costimulatory molecule or other ligand. Forexample, the agent may act intracellularly to stimulate a signalassociated with CD28 ligation. In one embodiment, the agent is anon-proteinaceous compound. As the agent used in the method is intendedto bypass the natural receptor:ligand stimulatory mechanism, the termagent is not intended to include a cell expressing a natural ligand.Natural ligands for CD28 include members of the B7 family of proteins,such as B7-1 (CD80) and B7-2 (CD86).

It is known that CD28 receptor stimulation leads to the production ofD-3 phosphoinositides in T cells and that inhibition of the activity ofphosphatidylinositol 3-kinase (PI3K) in a T cell can inhibit T cellresponses, such as lymphokine production and cellular proliferation.Protein tyrosine phosphorylation has also been shown to occur in T cellsupon CD28 ligation and it has been demonstrated that a protein tyrosinekinase inhibitor, herbimycin A, can inhibit CD28-induced IL-2 production(Vandenberghe, P. et al. (1992) J. Exp. Med. 175:951-960; Lu, Y. et al.(1992) J. Immunol. 149:24-29). Thus, to selectively expand a populationof CD4⁺ T cells, the CD28 receptor mediated pathway can be stimulated bycontacting T cells with an activator of PI3K or an agent whichstimulates protein tyrosine phosphorylation in the T cell, or both. Anactivator of PI3K can be identified based upon its ability to stimulateproduction of at least one D-3 phosphoinositide in a T cell. The term“D-3 phosphoinositide” is intended to include derivatives ofphosphatidylinositol that are phosphorylated at the D-3 position of theinositol ring and encompasses the compoundsphosphatidylinositol(3)-monophosphate (PtdIns(3)P),phosphatidylinositol(3,4)-bisphosphate (PtdIns(3,4)P₂), andphosphatidylinositol(3,4,5)-trisphosphate (PtdIns(3,4,5)P₃). Thus, inthe presence of a PI3K activator, the amount of a D-3 phosphoinositidein the T cell is increased relative to the amount of the D-3phosphoinositide in the T cell in the absence of the substance.Production of D-3 phosphoinositides (e.g., PtdIns(3)P, PtdIns(3,4)P₂and/or PtdIns(3,4,5)P₃) in a T cell can be assessed by standard methods,such as high pressure liquid chromatography or thin layerchromatography, as discussed above. Similarly, protein tyrosinephosphorylation can be stimulated in a T cell, for example, bycontacting the T cell with an activator of protein tyrosine kinases,such as pervanadate (see O'Shea, J. J. et al. (1992) Proc. Natl. Acad.Sci. USA 89:10306-103101; and Secrist, J. P. (1993) J. Biol. Chem.268:5886-5893). Alternatively, the T cell can be contacted with an agentwhich inhibits the activity of a cellular protein tyrosine phosphatase,such as CD45, to increase the net amount of protein tyrosinephosphorylation in the T cell. Any of these agents can be used to expandan activated population of CD4⁺ T cells in accordance with the methodsdescribed herein.

Techniques for Expansion of CD8⁺ T Cells

In order to induce proliferation and expand a population of CD8⁺ Tcells, an activated population of T cells is stimulated through a 27 kDaccessory molecule found on activated T cells and recognized by themonoclonal antibody ES5.2D8. As described in Example 9, a population ofCD8⁺ T cells was preferentially expanded by stimulation with an anti-CD3monoclonal antibody and the ES5.2D8 monoclonal antibody. The monoclonalantibody ES5.2D8 was produced by immunization of mice with activatedhuman blood lymphocytes and boosted with recombinant human CTLA4 proteinproduced in E. coli. The ES5.2D8 monoclonal antibody is of the IgG2bisotype and specifically binds to cells transfected with human CTLA4.Hybridomas producing CTLA4-specific antibody were identified byscreening by ELISA against human CTLA4 protein as well as bydifferential FACS against wild type CHO-DG44 cells vs. CHO-105A cells,which are transfected with the human CTLA4 gene. As shown in FIG. 7, theES5.2D8 clone reacts strongly with both activated human T cells andCHO-105A cells but not with CHO-DCA4 cells, indicating that it doesindeed bind to CTLA4. Immunoprecipitation of detergent lysates ofsurface labeled activated human T cells revealed that ES5.2D8 alsoreacts with a 27 kD cell surface protein (FIG. 8). A hybridoma whichproduces the monoclonal antibody ES5.2D8 was deposited on Jun. 4, 1993with the American Type Culture Collection at ATCC Deposit No. HB11374.

Accordingly, to expand a population of CD8+ T cells, an antibody, suchas monoclonal antibody ES5.2D8, or other antibody which recognizes thesame 27 kD ligand as monoclonal antibody ES5.2D8 can be used. Asdescribed in Example 10, the epitope recognized by the monoclonalantibody ES5.2D8 was identified by screening a phage display library(PDL). Antibodies which bind to the same epitope as the monoclonalantibody ES5.2D8 are within the scope of the invention. Such antibodiescan be produced by immunization with a peptide fragment including theepitope or with the native 27 kD antigen. The term “epitope,” as usedherein, refers to the actual structural portion of the antigen that isimmunologically bound by an antibody combining site. The term is alsoused interchangeably with “antigenic determinant”. A preferred epitopewhich is bound by an antibody or other ligand which is to be used tostimulate a CD8+ T cell population includes or encompasses, an aminoacid sequence:(Xaa₁)_(n)-Gly-Xaa₂-Trp-Leu-Xaa₃-Xaa₄-Asp(Glu)-(Xaa₅)_(n)(SEQ ID NO:5),wherein Xaa₄ may or may not be present, Xaa₁, Xaa₂, Xaa₃, Xaa₄ and Xaa₅are any amino acid residue and n=0-20, more preferably 0-10, even morepreferably 0-5, and most preferably 0-3. In a preferred embodiment, Xaa₂is Cys, Ile or Leu, Xaa₃ is Leu or Arg and Xaa₄, if present, is Arg, Proor Phe. As described in Example 10, the monoclonal antibody ES5.2D8,which specifically binds a 27 kD antigen on activated T cells was usedto screen a cDNA library from activated T cells to isolate a cloneencoding the antigen. Amino acid sequence analysis identified theantigen as CD9 (SEQ ID NO: 6). In the native human CD9 molecule, epitopedefined by phage display library screening is located at amino acidresidues 31-37 (i.e., G L W L R F D (SEQ ID NO: 7)). Accordingly, Xaa₁and Xaa₄ are typically additional amino acid residues found at eitherthe amino or carboxy side, or both the amino and carboxy sides, of thecore epitope in the human CD9 (the full-length amino acid sequence ofwhich is shown in SEQ ID NO: 6). It will be appreciated by those skilledin the art that in the native protein, additional non-contiguous aminoacid residues may also contribute to the conformational epitoperecognized by the antibody. Synthetic peptides encompassing the epitopecan be created which includes other amino acid residues flanking thecore six amino acid residues (i.e. Xaa can alternatively be other aminoacid residues than those found in the native CD9 protein). Theseflanking amino acid residues can function to alter the properties of theresulting peptide, for example to increase the solubility, enhance theimmunogenicity or promote dimerization of the resultant peptide. Whenthe peptide is to be used as an immunogen, one or more charged aminoacids (e.g. lysine, arginine) can be included to increase the solubilityof the peptide and/or enhance the immunogenicity of the peptide.Alternatively, cysteine residues can be included to increase thedimerization of the resulting peptide.

Other embodiments of the invention pertain to expansion of a populationof CD8⁺ T cells by use of an agent which acts intracellularly tostimulate a signal in the T cell mediated by ligation of CD9 or otherCD9-associated molecule. It is known that CD9 belongs to the TM4superfamily of cell surface proteins which span the membrane four times(Boucheix, C. et al. (1990) J. Biol. Chem. 266, 117-122 and Lanza, F. etal. (1990) J. Biol. Chem. 266, 10638-10645). Other members of the TM4superfamily include CD37, CD53, CD63 and TAPA-1. A role for CD9 ininteracting with GTP binding proteins has been suggested (Sechafer, J.G. and Shaw, A. R. E. (1991) Biochem. Biophys. Res. Commun 179,401-406). As used herein the term “agent” encompasses chemicals andother pharmaceutical compounds which stimulate a signal in a T cellwithout the requirement for an interaction between a T cell surfacereceptor and a ligand. Thus, this agent does not bind to theextracellular portion of CD9, but rather mimics or induces anintracellular signal (e.g., second messenger) associated with ligationof CD9 or a CD9-associated molecule by an appropriate ligand. Theligands described herein (e.g., monoclonal antibody ES5.2D8) can be usedto identify an intracellular signal(s) associated with T cell expansionmediated by contact of the CD9 antigen or CD9-associated molecule withan appropriate ligand (as described in the Examples) and examining theresultant intracellular signalling that occurs (e.g., protein tyrosinephosphorylation, calcium influx, activation of serine/threonine and/ortyrosine kinases, phosphatidyl inositol metabolism, etc.). An agentwhich enhances an intracellular signal associated with CD9 or aCD9-associated molecule can then be used to expand CD8⁺ T cells.Alternatively, agents (e.g., small molecules, drugs, etc.) can bescreened for their ability to inhibit or enhance T cell expansion usinga system such as that described in the Examples.

Techniques for Expansion of Antigen Specific T Cells

In yet another aspect of the invention, methods for expanding apopulation of antigen specific T cells are provided. To produce apopulation of antigen specific T cells, T cells are contacted with anantigen in a form suitable to trigger a primary activation signal in theT cell, i.e., the antigen is presented to the T cell such that a signalis triggered in the T cell through the TCR/CD3 complex. For example, theantigen can be presented to the T cell by an antigen presenting cell inconduction with an MHC molecule. An antigen presenting cell, such as a Bcell, macrophage, monocyte, dendritic cell, Langerhan cell, or othercell which can present antigen to a T cell, can be incubated with the Tcell in the presence of the antigen (e.g., a soluble antigen) such thatthe antigen presenting cell presents the antigen to the T cell.Alternatively, a cell expressing an antigen of interest can be incubatedwith the T cell. For example, a tumor cell expressing tumor-associatedantigens can be incubated with a T cell together to induce atumor-specific response. Similarly, a cell infected with a pathogen,e.g., a virus, which presents antigens of the pathogen can be incubatedwith a T cell. Following antigen specific activation of a population ofT cells, the cells can be expanded in accordance with the methods of theinvention. For example, after antigen specificity has been established,T cells can be expanded by culture with an anti-CD3 antibody and ananti-CD28 antibody according to the methods described herein.

Production of Antibodies and Coupling of Antibodies to Solid PhaseSurfaces

The term “antibody” as used herein refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site which specifically binds(immunoreacts with) an antigen, such as CD3, CD28. Structurally, thesimplest naturally occurring antibody (e.g., IgG) comprises fourpolypeptide chains, two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds. It has been shown that theantigen-binding function of an antibody can be performed by fragments ofa naturally-occurring antibody. Thus, these antigen-binding fragmentsare also intended to be designated by the term “antibody”. Examples ofbinding fragments encompassed within the term antibody include (i) anFab fragment consisting of the VL, VH, CL and CH1 domains; (ii) an Fdfragment consisting of the VH and CH1 domains; (iii) an Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody, (iv)a dAb fragment (Ward et al., (1989) Nature 341:544-546) which consistsof a VH domain; (v) an isolated complimentarity determining region(CDR); and (vi) an F(ab′)₂ fragment, a bivalent fragment comprising twoFab fragments linked by a disulfide bridge at the hinge region.Furthermore, although the two domains of the Fv fragment are coded forby separate genes, a synthetic linker can be made that enables them tobe made as a single protein chain (known as single chain Fv (scFv); Birdet al. (1988) Science 242:423-426; and Huston et al. (1988) PNAS85:5879-5883) by recombinant methods. Such single chain antibodies arealso encompassed within the term “antibody”. Preferred antibodyfragments for use in T cell expansion are those which are capable ofcrosslinking their target antigen, e.g., bivalent fragments such asF(ab′)₂ fragments. Alternatively, an antibody fragment which does notitself crosslink its target antigen (e.g., a Fab fragment) can be usedin conjunction with a secondary antibody which serves to crosslink theantibody fragment, thereby crosslinking the target antigen. Antibodiescan be fragmented using conventional techniques as described herein andthe fragments screened for utility in the same manner as described forwhole antibodies. An antibody of the invention is further intended toinclude bispecific and chimeric molecules having a desired bindingportion (e.g., CD28).

The language “a desired binding specificity for an epitope”, as well asthe more general language “an antigen binding site which specificallybinds (immunoreacts with)”, refers to the ability of individualantibodies to specifically immunoreact with a T cell surface molecule,e.g., CD28. That is, it refers to a non-random binding reaction betweenan antibody molecule and an antigenic determinant of the T cell surfacemolecule. The desired binding specificity is typically determined fromthe reference point of the ability of the antibody to differentiallybind the T cell surface molecule and an unrelated antigen, and thereforedistinguish between two different antigens, particularly where the twoantigens have unique epitopes. An antibody which binds specifically to aparticular epitope is referred to as a “specific antibody”.

“Antibody combining site”, as used herein, refers to that structuralportion of an antibody molecule comprised of a heavy and light chainvariable and hypervariable regions that specifically binds (immunoreactswith) antigen. The term “immunoreact” or “reactive with” in its variousforms is used herein to refer to binding between an antigenicdeterminant-containing molecule and a molecule containing an antibodycombining site such as a whole antibody molecule or a portion thereof.

Although soluble forms of antibodies may be used to activate T cells, itis preferred that the anti-CD3 antibody be immobilized on a solid phasesurface (e.g., beads). An antibody can be immobilized directly orindirectly by, for example, by a secondary antibody, to a solid surface,such as a tissue culture flask or bead. As an illustrative embodiment,the following is a protocol for immobilizing an anti-CD3 antibody onbeads. It should be appreciated that the same protocol can be used toimmobilize other antibodies or fragments thereof (e.g., an anti-CD28antibody), and Ig fusion proteins, such as B7Ig fusion proteins, tobeads.

Protocols

-   -   I. Pre-absorbing Goat anti-mouse IgG with OKT-3        -   A) BioMag Goat anti-Mouse IgG (Advanced Magnetics, Inc.,            catalog number 8-4340D) is incubated with at least 200 μg of            OKT-3 per 5×10⁸ magnetic particles in PBS for 1 hour at 5°            C.        -   B) Particles are washed three time in PBS with the aid of a            magnetic separation unit.        -   Note: Advanced Magnetics also has an anti-Human CD3 directly            conjugated (Catalog number 8-4703N) which will induce T-cell            stimulation.    -   II. Pre-labeling Lymphocytes with OKT-3        -   A) 1×10⁶ cells (PBMC) are incubated in PBS with 10 μg/ml of            OKT-3 for 15 minutes at room temperature.        -   B) Cells are washed twice with PBS.    -   III. Binding Magnetic Particles to PBMC for Stimulation        -   A) PBMC surface labeled with OKT-3 are cultured with Goat            anti-Mouse IgG (see above) at one bead per cell following a            30 minute incubation at 20° C. with gentle agitation.        -   B) Goat anti-Mouse IgG beads which were previously absorbed            to OKT-3 are incubated with PBMC (1:1) for 30 minutes at            20° C. with gentle agitation and cultured.    -   IV. Binding Magnetic Particles to PBMC for Separation Same as        above (Part III) except the bead to cell ratio is increased to        20:1 rather than 1:1.

Alternatively, antibodies can be coupled to a solid phase surface, e.g.,beads by crosslinking via covalent modification using tosyl linkage. Inone method, an antibody such as OKT3 is in 0.05M borate buffer, pH 9.5and added to tosyl activated magnetic immunobeads (Dynal Inc., GreatNeck, N.Y.) according to the manufacturer's instructions. After a 24 hrincubation at 22° C., the beads are collected and washed extensively. Itis not mandatory that immunomagnetic beads be used, as other methods arealso satisfactory.

To practice the method of the invention, a source of T cells is obtainedfrom a subject. The term subject is intended to include living organismsin which an immune response can be elicited, e.g., mammals. Examples ofsubjects include humans, dogs, cats, mice, rats, and transgenic speciesthereof. T cells can be obtained from a number of sources, includingperipheral blood leukocytes, bone marrow, lymph node tissue, spleentissue, and tumors. Preferably, peripheral blood leukocytes are obtainedfrom an individual by leukopheresis. To isolate T cells from peripheralblood leukocytes, it may be necessary to lyse the red blood cells andseparate peripheral blood leukocytes from monocytes by, for example,centrifugation through a PERCOLL™ gradient. A specific subpopulation ofT cells, such as CD28⁺, CD4⁺, CD8⁺, CD28RA⁺, and CD28RO⁺T cells, can befurther isolated by positive or negative selection techniques. Forexample, negative selection of a T cell population can be accomplishedwith a combination of antibodies directed to surface markers unique tothe cells negatively selected. A preferred method is cell sorting vianegative magnetic immunoadherence which utilizes a cocktail ofmonoclonal antibodies directed to cell surface markers present on thecells negatively selected. For example, to isolate CD4⁺ cells, amonoclonal antibody cocktail typically includes antibodies to CD14,CD20, CD11b, CD16, HLA-DR, and CD8. Additional monoclonal antibodycocktails are provided in Table 1.

The process of negative selection results in an essentially homogenouspopulation of CD28⁺, CD4⁺ or CD8⁺ T cells. The T cells can be activatedas described herein, such as by contact with a anti-CD3 antibodyimmobilized on a solid phase surface or an anti-CD2 antibody, or bycontact with a protein kinase C activator (e.g., bryostatin) inconjunction with a calcium ionophore. To stimulate an accessory moleculeon the surface of the T cells, a ligand which binds the accessorymolecule is employed. For example, a population of CD4⁺ cells can becontacted with an anti-CD3 antibody and an anti-CD28 antibody, underconditions appropriate for stimulating proliferation of the T cells.Similarly, to stimulate proliferation of CD8⁺ T cells, an anti-CD3antibody and the monoclonal antibody ES5.2D8 can be used. Conditionsappropriate for T cell culture include an appropriate media (e.g.,Minimal Essential Media or RPMI Media 1640) which may contain factorsnecessary for proliferation and viability, including animal serum (e.g.,fetal bovine serum) and antibiotics (e.g., penicillin streptomycin). TheT cells are maintained under conditions necessary to support growth, forexample an appropriate temperature (e.g., 37° C.) and atmosphere (e.g.,air plus 5% CO₂).

The primary activation signal and the costimulatory signal for the Tcell can be provided by different protocols. For example, the agentsproviding each signal can be in solution or coupled to a solid phasesurface. When coupled to a solid phase surface, the agents can becoupled to the same solid phase surface (i.e., in “cis” formation) or toseparate surfaces (i.e., in “trans” formation). Alternatively, one agentcan be coupled to a solid phase surface and the other agent in solution.In one embodiment, the agent providing the costimulatory signal is boundto a cell surface and the agent providing the primary activation signalis in solution or coupled to a solid phase surface. In a preferredembodiment, the two agents are coupled to beads, either to the samebead, i.e., in “cis”, or to separate beads, i.e., in “trans”.Alternatively, the agent providing the primary activation signal is ananti-CD3 antibody and the agent providing the costimulatory signal is ananti-CD28 antibody; both agents are coupled to the same beads. In thisembodiment, it has been determined that the optimal ratio of eachantibody bound to the beads for CD4⁺ T cell expansion and T cell growthfor up to at least 50 days is a 1:1 ratio. However, ratios from 1:9 to9:1 can also be used to stimulate CD2⁺ T cell expansion. The ratio ofanti-CD3 and anti-CD28 coated (with a ratio of 1:1 of each antibody)beads to T cells that yield T cell expansion can vary from 1:3 to 3:1,with the optimal ratio being 3:1 beads per T cell. Moreover, it has beendetermined that when T cells are expanded under these conditions, theyremain polyclonal.

To maintain long term stimulation of a population of T cells followingthe initial activation and stimulation, it is necessary to separate theT cells from the activating stimulus (e.g., the anti-CD3 antibody) aftera period of exposure. The T cells are maintained in contact with theco-stimulatory ligand throughout the culture term. The rate of T cellproliferation is monitored periodically (e.g., daily) by, for example,examining the size or measuring the volume of the T cells, such as witha COULTER COUNTER®. A resting T cell has a mean diameter of about 6.8microns. Following the initial activation and stimulation and in thepresence of the stimulating ligand, the T cell mean diameter willincrease to over 12 microns by day 4 and begin to decrease by about day6. When the mean T cell diameter decreases to approximately 8 microns,the T cells are reactivated and restimulated to induce furtherproliferation of the T cells. Alternatively, the rate of T cellproliferation and time for T cell restimulation can be monitored byassaying for the presence of cell surface molecules, such as B7-1, B7-2,which are induced on activated T cells. As described in Example 5, itwas determined that CD4⁺ T cells do not initially express the B7-1receptor, and that with culture, expression is induced. Further, theB7-1 expression was found to be transient, and could be re-induced withrepeated anti-CD3 restimulation. Accordingly, cyclic changes in B7-1expression can be used as a means of monitoring T cell proliferation;where decreases in the level of B7-1 expression, relative to the levelof expression following an initial or previous stimulation or the levelof expression in an unstimulated cell, indicates the time forrestimulation.

For inducing long term stimulation of a population of CD4⁺ or CD8⁺ Tcells, it may be necessary to reactivate and restimulate the T cellswith a anti-CD3 antibody and an anti-CD28 antibody or monoclonalantibody ES5.2D8 several times to produce a population of CD4⁺ or CD8⁺cells increased in number from about 10- to about 1,000-fold theoriginal T cell population. Using this methodology, it is possible toget increases in a T cell population of from about 100- to about100,000-fold an original resting T cell population. Moreover, asdescribed in Example 6, T cells expanded by the method of the inventionsecrete high levels of cytokines (e.g., IL-2, IFNλ, IL-4, GM-CSF andTNFα) into the culture supernatants. For example, as compared tostimulation with IL-2, CD4⁺ T cells expanded by use of anti-CD3 andanti-CD28 costimulation secrete high levels of GM-CSF and TNFα into theculture medium. These cytokines can be purified from the culturesupernatants or the supernatants can be used directly for maintainingcells in culture. Similarly, the T cells expanded by the method of theinvention together with the culture supernatant and cytokines can beadministered to support the growth of cells in vivo. For example, inpatients with tumors, T cells can be obtained from the individual,expanded in vitro and the resulting T cell population and supernatant,including cytokines such as TNFα, can be readministered to the patientto augment T cell growth in vivo.

Although the antibodies used in the methods described herein can bereadily obtained from public sources, such as the ATCC, antibodies to Tcell surface accessory molecules, the CD3 complex, or CD2 can beproduced by standard techniques. Methodologies for generating antibodiesfor use in the methods of the invention are described in further detailbelow.

Specific Methodology for Antibody Production

A. The Immunogen. The term “immunogen” is used herein to describe acomposition containing a peptide or protein as an active ingredient usedfor the preparation of antibodies against an antigen (e.g., CD3, CD28).When a peptide or protein is used to induce antibodies it is to beunderstood that the peptide can be used alone, or linked to a carrier asa conjugate, or as a peptide polymer.

To generate suitable antibodies, the immunogen should contain aneffective, immunogenic amount of a peptide or protein, optionally as aconjugate linked to a carrier. The effective amount of peptide per unitdose depends, among other things, on the species of animal inoculated,the body weight of the animal and the chosen immunization regimen as iswell known in the art. The immunogen preparation will typically containpeptide concentrations of about 10 micrograms to about 500 milligramsper immunization dose, preferably about 50 micrograms to about 50milligrams per dose. An immunization preparation can also include anadjuvant as part of the diluent. Adjuvants such as complete Freund'sadjuvant (CFA), incomplete Freund's adjuvant (IFA) and alum arcmaterials well known in the art, and are available commercially fromseveral sources.

Those skilled in the art will appreciate that, instead of using naturaloccurring forms of the antigen (e.g., CD3, CD28) for immunization,synthetic peptides can alternatively be employed towards whichantibodies can be raised for use in this invention. Both soluble andmembrane bound forms of the protein or peptide fragments are suitablefor use as an immunogen and can also be isolated by immunoaffinitypurification as well. A purified form of protein, such as may beisolated as described above or as known in the art, can itself bedirectly used as an immunogen, or alternatively, can be linked to asuitable carrier protein by conventional techniques, including bychemical coupling means as well as by genetic engineering using a clonedgene of the protein. The purified protein can also be covalently ornoncovalently modified with non-proteinaceous materials such as lipidsor carbohydrates to enhance immunogenecity or solubility. Alternatively,a purified protein can be coupled with or incorporated into a viralparticle, a replicating virus, or other microorganism in order toenhance immunogenicity. The protein may be, for example, chemicallyattached to the viral particle or microorganism or an immunogenicportion thereof.

In an illustrative embodiment, a purified CD28 protein, or a peptidefragment thereof (e.g., produced by limited proteolysis or recombinantDNA techniques) is conjugated to a carrier which is immunogenic inanimals. Preferred carriers include proteins such as albumins, serumproteins (e.g., globulins and lipoproteins), and polyamino acids.Examples of useful proteins include bovine serum albumin, rabbit serumalbumin, thyroglobulin, keyhole limpet hemocyanin, egg ovalbumin andbovine gamma-globulins. Synthetic polyamino acids such as polylysine orpolyarginine are also useful carriers. With respect to the covalentattachment of CD28 protein or peptide fragments to a suitableimmunogenic carrier, there are a number of chemical cross-linking agentsthat are known to those skilled in the art. Preferred cross-linkingagents are heterobifunctional cross-linkers, which can be used to linkproteins in a stepwise manner. A wide variety of heterobifunctionalcross-linkers are known in the art, including succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-succinimidyl(4-iodoacetyl) aminobenzoate (SIAB), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC);4-succinimidyl-oxycarbonyl-a-methyl-a-(2-pyridyldithio)-tolune (SMPT),N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), succinimidyl6-[3-(2-pyridyldithio) propionate] hexanoate (LC-SPDP).

It may also be desirable to simply immunize an animal with whole cellswhich express a protein of interest (e.g., CD28) on their surface.Various cell lines can be used as immunogens to generate monoclonalantibodies to an antigen, including, but not limited to T cells. Forexample, peripheral blood T cells can be obtained from a subject whichconstituitively express CD28, but can be activated in vitro withanti-CD3 antibodies, PHA or PMA. Alternatively, an antigen specific(e.g., alloreactive) T cell clone can be activated to express CD28 bypresentation of antigen, together with a costimulatory signal, to the Tcell. Whole cells that can be used as immunogens to produce CD28specific antibodies also include recombinant transfectants. For example,COS and CHO cells can be reconstituted by transfection with a CD28 cDNAto produce cells expressing CD28 on their surface. These transfectantcells can then be used as immunogens to produce anti-CD28 antibodies.Other examples of transfectant cells are known, particularly eukaryoticcells able to glycosylate the. CD28 protein, but any procedure thatworks to express transfected CD28 genes on the cell surface could beused to produce the whole cell immunogen.

Alternative to a CD28-expressing cell or an isolated CD28 protein,peptide fragments of CD28 or other surface antigen such as CD9 can beused as immunogens to generate antibodies. For example, the CD9 epitopebound by the ES5.2D8 monoclonal antibody comprises an amino acidsequence: (Xaa₁)_(n)-Gly-Xaa₂-Trp-Leu-Xaa₃-Xaa₄-Asp(Glu)-(Xaa₅)_(n)(SEQID NO: 5), wherein Xaa₄may or may not be present, Xaa₁, Xaa₂, Xaa₃, Xaa₄and Xaa₅ are any amino acid residue and n=0-20, more preferably 0-10,even more preferably 0-5, and most preferably 0-3. In a preferredembodiment, Xaa₂ is Cys, Ile or Leu, Xaa₃ is Leu or Arg and Xaa₄, ifpresent, is Arg, Pro or Phe. Thus, a peptide having the amino acidsequence of SEQ ID NO: 5 can be used as an immunogen. Accordingly, theinvention further encompasses an isolated CD9 peptide comprising anamino acid sequence:(Xaa₁)_(n)-Gly-Xaa₂-Trp-Leu-Xaa₃-Xaa₄-Asp(Glu)-(Xaa₅)_(n)(SEQ ID NO: 5),wherein Xaa₄ may or may not be present, Xaa₁, Xaa₂, Xaa₃, Xaa₄ and Xaa₅are any amino acid residue and n=0-20, more preferably 0-10, even morepreferably 0-5, and most preferably 0-3. In a preferred embodiment, Xaa₂is Cys, Ile or Leu, Xaa₃ is Leu or Arg and Xaa₄, if present, is Arg, Proor Phe. Alternatively, it has been found that the ES5.2D8 monoclonalantibody cross-reacts with a number of other peptide sequences(determined by phage display technology as described in Example 3).Examples of these other peptide sequences are shown below:

-   -   2D8#2(SEQ ID NO:8) H Q F C D H W G C W L L R E T H I F T P    -   2D8#4(SEQ ID NO:8) H Q F C D H W G C W L L R E T H I F T P    -   2D8#10(SEQ ID NO:8) H Q F C D H W G C W L L R E T H I F T P    -   2D8#6(SEQ ID NO:9) L R L V L E D P G I W L R P D Y F F P A    -   phage 2D8#2,4, 10 (SEQ ID NO: 10) G C W L L R E    -   phage 2D8#6 (SEQ ID NO: 11) G I W L R P D    -   CD9 sequence (SEQ ID NO: 7) G L W L R F D        Any of these peptides, or other peptides containing a stretch of        seven amino acids bracketed in bold type (representing the        epitope bound by the antibody) possibly flanked by alternative        amino acid residues, can also be used as immunogens to produce        an antibody for use in the methods of the invention and are        encompassed by the invention. For use as immunogens, peptides        can be modified to increase solubility and/or enhance        immunogenicity as described above.

B. Polyclonal Antibodies. Polycolonal antibodies to a purified proteinor peptide fragment thereof can generally be raised in animals bymultiple subcutaneous (sc) or intraperitoneal (ip) injections of anappropriate immunogen, such as the extracellular domain of the protein,and an adjuvant. A polyclonal antisera can be produced, for example, asdescribed in Lindsten, T. et al. (1993) J. Immunol. 151:3489-3499. In anillustrative embodiment, animals are typically immunized against theimmunogenic protein, peptide or derivative by combining about 1 μg to 1mg of protein with Freund's complete adjuvant and injecting the solutionintradermally at multiple sites. One month later the animals are boostedwith ⅕ to {fraction (1/10)} the original amount of immunogen in Freund'scomplete adjuvant (or other suitable adjuvant) by subcutaneous injectionat multiple sites. Seven to 14 days later, the animals are bled and theserum is assayed for anti-protein or peptide titer (e.g., by ELISA).Animals are boosted until the titer plateaus. Also, aggregating agentssuch as alum can be used to enhance the immune response.

Such mammalian-produced populations of antibody molecules are referredto as “polyclonal” because the population comprises antibodies withdiffering immunospecificities and affinities for the antigen. Theantibody molecules are then collected from the mammal (e.g., from theblood) and isolated by well known techniques, such as protein Achromatography, to obtain the IgG fraction. To enhance the specificityof the antibody, the antibodies may be purified by immunoaffinitychromatography using solid phase-affixed immunogen. The antibody iscontacted with the solid phase-affixed immunogen for a period of timesufficient for the immunogen to immunoreact with the antibody moleculesto form a solid phase-affixed immunocomplex. The bound antibodies areseparated from the complex by standard techniques.

C. Monoclonal Antibodies. The term “monoclonal antibody” or “monoclonalantibody-composition”, as used herein, refers to a population ofantibody molecules that contain only one species of an antigen bindingsite capable of immunoreacting with a particular epitope of an antigen.A monoclonal antibody composition thus typically displays a singlebinding affinity for a particular protein with which it immunoreacts.Preferably, the monoclonal antibody used in the subject method isfurther characterized as immunoreacting with a protein derived fromhumans.

Monoclonal antibodies useful in the methods of the invention aredirected to an epitope of an antigen(s) on T cells, such that complexformation between the antibody and the antigen (also referred to hereinas ligation) induces stimulation and T cell expansion. A monoclonalantibody to an epitope of an antigen (e.g., CD3, CD28) can be preparedby using a technique which provides for the production of antibodymolecules by continuous cell lines in culture. These include but are notlimited to the hybridoma technique originally described by Kohler andMilstein (1975, Nature 256:495-497), and the more recent human B cellhybridoma technique (Kozbor et al. (1983) Immunol Today 4:72),EBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96), and trioma techniques.Other methods which can effectively yield monoclonal antibodies usefulin the present invention include phage display techniques (Marks et al.(1992) J Biol Chem 16007-16010).

In one embodiment, the antibody preparation applied in the subjectmethod is a monoclonal antibody produced by a hybridoma cell line.Hybridoma fusion techniques were first introduced by Kohler and Milstein(Kohler et al. Nature (1975) 256:495-97; Brown et al. (1981)J Immunol127:539-46; Brown et al. (1980) J Biol Chem 255:4980-83; Yeh et al.(1976) PNAS 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75).Thus, the monoclonal antibody compositions of the present invention canbe produced by the following method, which comprises the steps of:

(a) Immunizing an animal with a protein (e.g., CD28) or peptide thereof.The immunization is typically accomplished by administering theimmunogen to an immunologically competent mammal in an immunologicallyeffective amount, i.e., an amount sufficient to produce an immuneresponse. Preferably, the mammal is a rodent such as a rabbit, rat ormouse. The mammal is then maintained for a time period sufficient forthe mammal to produce cells secreting antibody molecules thatimmunoreact with the immunogen. Such immunoreaction is detected byscreening the antibody molecules so produced for immunoreactivity with apreparation of the immunogen protein. Optionally, it may be desired toscreen the antibody molecules with a preparation of the protein in theform in which it is to be detected by the antibody molecules in anassay, e.g., a membrane-associated form of the antigen (e.g., CD28).These screening methods are well known to those of skill in the art,e.g., enzyme-linked immunosorbent assay (ELISA) and/or flow cytometry.

(b) A suspension of antibody-producing cells removed from each immunizedmammal secreting the desired antibody is then prepared. After asufficient time, the mouse is sacrificed and somatic antibody-producinglymphocytes are obtained. Antibody-producing cells may be derived fromthe lymph nodes, spleens and peripheral blood of primed animals. Spleencells are preferred, and can be mechanically separated into individualcells in a physiologically tolerable medium using methods well known inthe art. Mouse lymphocytes give a higher percentage of stable fusionswith the mouse myelomas described below. Rat, rabbit and frog somaticcells can also be used. The spleen cell chromosomes encoding desiredimmunoglobulins are immortalized by fusing the spleen cells with myelomacells, generally in the presence of a fusing agent such as polyethyleneglycol (PEG). Any of a number of myeloma cell lines may be used as afusion partner according to standard techniques; for example, theP3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. Thesemyeloma lines are available from the American Type Culture Collection(ATCC), Rockville, Md.

The resulting cells, which include the desired hybridomas, are thengrown in a selective medium, such as HAT medium, in which unfusedparental myeloma or lymphocyte cells eventually die. Only the hybridomacells survive and can be grown under limiting dilution conditions toobtain isolated clones. The supernatants of the hybridomas are screenedfor the presence of antibody of the desired specificity, e.g., byimmunoassay techniques using the antigen that has been used forimmunization. Positive clones can then be subcloned under limitingdilution conditions and the monoclonal antibody produced can beisolated. Various conventional methods exist for isolation andpurification of the monoclonal antibodies so as to free them from otherproteins and other contaminants. Commonly used methods for purifyingmonoclonal antibodies include ammonium sulfate precipitation, ionexchange chromatography, and affinity chromatography (see, e.g., Zola etal. in Monoclonal Hybridoma Antibodies: Techniques And Applications,Hurell (ed.) pp. 51-52 (CRC Press 1982)). Hybridomas produced accordingto these methods can be propagated in vitro or in vivo (in ascitesfluid) using techniques known in the art.

Generally, the individual cell line may be propagated in vitro, forexample in laboratory culture vessels, and the culture medium containinghigh concentrations of a single specific monoclonal antibody can beharvested by decantation, filtration or centrifugation. Alternatively,the yield of monoclonal antibody can be enhanced by injecting a sampleof the hybridoma into a histocompatible animal of the type used toprovide the somatic and myeloma cells for the original fusion. Tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid develop in the injected animal. The body fluids of the animal,such as ascites fluid or serum, provide monoclonal antibodies in highconcentrations. When human hybridomas or EBV-hybridomas are used, it isnecessary to avoid rejection of the xenograft injected into animals suchas mice. Immunodeficient or nude mice may be used or the hybridoma maybe passaged first into irradiated nude mice as a solid subcutaneoustumor, cultured in vitro and then injected intraperitoneally intopristane primed, irradiated nude mice which develop ascites tumorssecreting large amounts of specific human monoclonal antibodies.

Media and animals useful for the preparation of these compositions areboth well known in the art and commercially available and includesynthetic culture media, inbred mice and the like. An exemplarysynthetic medium is Dulbecco's minimal essential medium (DMEM; Dulbeccoet al. (1959) Virol. 8:396) supplemented with 4.5 gm/l glucose, 20 mMglutamine, and 20% fetal caf serum. An exemplary inbred mouse strain isthe Balb/c.

D. Combinatorial Antibodies. Monoclonal antibody compositions of theinvention can also be produced by other methods well known to thoseskilled in the art of recombinant DNA technology. An alternative method,referred to as the “combinatorial antibody display” method, has beendeveloped to identify and isolate antibody fragments having a particularantigen specificity, and can be utilized to produce monoclonalantibodies (for descriptions of combinatorial antibody display see e.g.,Sastry et al. (1989) PNAS 86:5728; Huse et al. (1989) Science 246:1275;and Orlandi et al. (1989) PNAS 86:3833). After immunizing an animal withan appropriate immunogen (e.g., CD3, CD28) as described above, theantibody repertoire of the resulting B-cell pool is cloned. Methods aregenerally known for directly obtaining the DNA sequence of the variableregions of a diverse population of immunoglobulin molecules by using amixture of oligomer primers and PCR. For instance, mixed oligonucleotideprimers corresponding to the 5′ leader (signal peptide) sequences and/orframework 1 (FR1) sequences, as well as primer to a conserved 3′constant region primer can be used for PCR amplification of the heavyand light chain variable regions from a number of murine antibodies(Larrick et al. (1991) Biotechniques 11:152-156). A similar strategy canalso been used to amplify human heavy and light chain variable regionsfrom human antibodies (Larrick et al. (1991) Methods: Companion toMethods in Enzymology 2:106-110).

In an illustrative embodiment, RNA is isolated from activated B cellsof, for example, peripheral blood cells, bone marrow, or spleenpreparations, using standard protocols (e.g., U.S. Pat. No. 4,683,202;Orlandi, et al. PNAS(1989) 86:3833-3837; Sastry et al., PNAS (1989)86:5728-5732; and Huse et al. (1989) Science 246:1275-1281.)First-strand cDNA is synthesized using primers specific for the constantregion of the heavy chain(s) and each of the κ and λ light chains, aswell as primers for the signal sequence. Using variable region PCRprimers, the variable regions of both heavy and light chains areamplified, each alone or in combinantion, and ligated into appropriatevectors for further manipulation in generating the display packages.Oligonucleotide primers useful in amplification protocols may be uniqueor degenerate or incorporate inosine at degenerate positions.Restriction endonuclease recognition sequences may also be incorporatedinto the primers to allow for the cloning of the amplified fragment intoa vector in a predetermined reading frame for expression.

The V-gene library cloned from the immunization-derived antibodyrepertoire can be expressed by a population of display packages,preferably derived from filamentous phage, to form an antibody displaylibrary. Ideally, the display package comprises a system that allows thesampling of very large variegated antibody display libraries, rapidsorting after each affinity separation round, and easy isolation of theantibody gene from purified display packages. In addition tocommercially available kits for generating phage display libraries(e.g., the Pharmacia Recombinant Phage Antibody System, catalog no.27-9400-01; and the Stratagene SurfZAP™ phage display kit, catalog no.240612), examples of methods and reagents particularly amenable for usein generating a variegated antibody display library can be found in, forexample, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al.International Publication No. WO 92/18619; Dower et al. InternationalPublication No. WO 91/17271; Winter et al. International Publication WO92/20791; Markland et al. International Publication No. WO 92/15679;Breitling et al. International Publication WO 93/01288; McCafferty etal. International Publication No. WO 92/101047; Garrard et al.International Publication No. WO 92/09690; Ladner et al. InternationalPublication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse etal. (1989) Science 246:1275-1281; Griffths et al. (1993) EMBOJ12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson etal. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:357-3580;Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al.(1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS88:7978-7982.

In certain embodiments, the V region domains of heavy and light chainscan be expressed on the same polypeptide, joined by a flexible linker toform a single-chain Fv fragment, and the scFV gene subsequently clonedinto the desired expression vector or phage genome. As generallydescribed in McCafferty et al., Nature (1990) 348:552-554, completeV_(H) and V_(L) domains of an antibody, joined by a flexible (Gly₄-Ser)₃linker can be used to produce a single chain antibody which can renderthe display package separable based on antigen affinity. Isolated scFVantibodies immunoreactive with the antigen can subsequently beformulated into a pharmaceutical preparation for use in the subjectmethod.

Once displayed on the surface of a display package (e.g., filamentousphage), the antibody library is screened with the protein, or peptidefragment thereof, to identify and isolate packages that express anantibody having specificity for the protein. Nucleic acid encoding theselected antibody can be recovered from the display package (e.g., fromthe phage genome) and subcloned into other expression vectors bystandard recombinant DNA techniques.

E. Hybridomas and Methods of Preparation. Hybridomas useful in thepresent invention are those characterized as having the capacity toproduce a monoclonal antibody which will specifically immunoreact withan antigen of interest (e.g., CD3, CD28). Methods for generatinghybridomas that produce, e.g., secrete, antibody molecules having adesired immunospecificity, e.g., having the ability to immunoreact withthe CD28 antigen, and/or an identifiable epitope of CD28 are well knownin the art Particularly applicable is the hybridoma technology describedby Niman et al. (1983) PNAS 8:4949-4953; and by Galfre et al. (1981)Meth. Enzymol. 73:3-46.

Uses of the Methods of the Invention

The method of this invention can be used to selectively expand apopulation of CD28⁺, CD4⁺, CD8⁺, CD28RA⁺, or CD28RO⁺T cells for use inthe treatment of infectious disease, cancer and immunotherapy. As aresult of the method described herein, a population of T cells which ispolyclonal with respect to antigen reactivity, but essentiallyhomogeneous with respect to either CD4⁺ or CD8⁺ can be produced. Inaddition, the method allows for the expansion of a population of T cellsin numbers sufficient to reconstitute an individual's total CD4⁺ or CD8⁺T cell population (the population of lymphocytes in an individual isapproximately 10¹¹). The resulting T cell population can be geneticallytransduced and used for immunotherapy or can be used in methods of invitro analyses of infectious agents. For example, a population oftumor-infiltrating lymphocytes can be obtained from an individualafflicted with cancer and the T cells stimulated to proliferate tosufficient numbers. The resulting T cell population can be geneticallytransduced to express tumor necrosis factor (TNF) or other factor andrestored to the individual.

One particular use for the CD4⁺ T cells expanded by the method of theinvention is in the treatment of HIV infection in an individual.Prolonged infection with HIV eventually results in a marked decline inthe number of CD4⁺ T lymphocytes. This decline, in turn, causes aprofound state of immunodeficiency, rendering the patient susceptible toan array of life threatening opportunistic infections. Replenishing thenumber of CD4⁺ T cells to normal levels may be expected to restoreimmune function to a significant degree. Thus, the method describedherein provides a means for selectively expanding CD4⁺ T cells tosufficient numbers to reconstitute this population in an HIV infectedpatient. It may also be necessary to avoid infecting the T cells duringlong-term stimulation or it may desirable to render the T cellspermanently resistant to HIV infection. There are a number of techniquesby which T cells may be rendered either resistant to HIV infection orincapable of producing virus prior to restoring the T cells to theinfected individual. For example, one or more anti-retroviral agents canbe cultured with CD4⁺ T cells prior to expansion to inhibit HIVreplication or viral production (e.g., drugs that target reversetranscriptase and/or other components of the viral machinery, see e.g.,Chow et al. (1993) Nature 361, 650-653).

Several methods can be used to genetically transduce T cells to producemolecules which inhibit HIV infection or replication. For example, inone embodiment, T cells can be genetically transduced to producetransdominant inhibitors, which are mutated, nonfunctional forms ofnormal HIV gene products. Transdominant inhibitors function tooligomerize or compete for binding with the wild type HIV proteins.Several transdominant inhibitors have been derived from HIV proteinsincluding tat, rev, and gag. The function of tat is to enhance thetranscription of viral genes by binding to the trans activation responseelement (tar) found in the promoter region of most HIV genes. Rev,through binding to the rev response element (RRE) found at the 59 end ofunspliced HIV transcripts, facilitates the transport of unprocessed mRNAfrom the nucleus to the cytoplasm for packaging into virions. Gag isfirst synthesized as a single polypeptide and subsequently cleaved by avirus-encoded protease to yield three structural proteins, p15, p17, andp24. Transdominant inhibitors derived from these gene products have beendemonstrated to inhibit infection of cells cultured with lab pet HIVisolates. One example of a transdominant inhibitor which appears to actby forming nonfunctional multimers with wild-type Rev is RevM10. RevM10construct has blocked infection of CEM cells by HTLV-IIIB for up to 28days (Malim et al. JEM 176:1197, Bevec et al. PNAS 89:9870). In thesestudies, RevM10 failed to demonstrate adverse effect on normal T cellfunction as judged by the criteria of growth rate and IL-2 secretion.

In another approach T cells can be transduced to produce molecules knownas “molecular decoys” which are binding elements for viral proteinscritical to replication or assembly, such as TAR. High levelretrovirus-mediated expression of TAR in CEM SS cells has been found toeffectively block the ARV-2 HIV isolate, as measured by RT assay(Sullenger et al. Cell 63:601). Importantly, it also blocked SIV(SIVmac251) infection, suggesting that inhibition of HIV infection withmolecular decoys may be generally applicable to various isolates andthereby alleviate the problem of hypervariability. Further, it has beendemonstrated that TAR expression has no discernible effects on cellviability (Sullenger et al. J. Virol. 65:6811). Another “moleculardecoy” which T cells can be transduced to produce is a soluble CD4tagged at the carboxy terminus with a KDEL (lysine-asparticacid-glutamic acid-leucine) sequence, a signal for ER retention(Buonocore and Rose, PNAS 90:2695)(Nature 345:625). The sCD4-KDEL geneexpression is driven by the HIV LTR. H9 cells transduced with thesCD4-KDEL construct show up regulation of expression of intracellularCD4 upon HIV infection. This strategy effectively blocked production ofHIV MN for up to 60 days post infection. The proposed advantage of thisinhibitor is that the virus should not be able to escape it's effect bymutating because CD4 binding is essential for HIV infectivity.

T cells can also be transduced to express antisense molecules andribozyme which block viral replication or infection. Viral replicationcan be inhibited with a variety of antisense strategies. One particularribozyme which cleaves HIV integrase (Sioud and Drlica. PNAS 88:7303),has been developed and may offer an approach to blocking infection asopposed to merely viral production.

Another approach to block HIV infection involves transducing T cellswith HIV-regulated toxins. Two examples of this type of approach are thediphtheria toxin A gene (Harrison et al. AIDS Res. Hum. Retro. 8:39) andthe herpes simplex virus type 1 thymidine kinase gene (HSV TK) (Carusoand Klatzmann, PNAS 89:182). In both cases, transcription was under thecontrol of HIV regulatory sequences. While the diphtheria toxin isitself toxic, the HSV TK requires the addition of acyclovir to killinfected cells. For example the use of HSV TK followed by the additionof 10 μm acyclovir for 17 days totally blocks HIV infection of HUT 78cells for up to 55 days of culture.

It has been demonstrated that when CD4⁺ T cells from an HIV infectedindividual are stimulated with anti-CD3 and anti-CD28 antibodiesattached to a solid phase support, such as beads, the cell cultureproliferates exponentially and the amount of HIV particles produced issignificantly reduced as compared to conventional methods forstimulating T cells, such as with PHA and IL-2 (see Example 16). Thus,when CD4⁺ T cells from an HIV infected individual are expanded ex vivowith anti-CD3 and anti-CD28 on a solid phase surface, the presence ofanti-retroviral agents may not be required in the culture to limitreplication of HIV. Since anti-retroviral drugs have toxic effects oncells, no anti-retroviral agent or reduced amounts of these agents tothe T cell culture will result in expansion to higher cell numbers.Thus, in a preferred embodiment of the invention, CD4⁺ T cells from anHIV infected individual are isolated, expanded ex vivo with anti-CD3 andanti-CD28 antibody coated beads (preferably, at a ratio of 3 beads per Tcell) in the absence of or in the presence of reduced amounts ofanti-retroviral agents and readministered to the individual.

The invention also provides for in vivo expansion of CD4⁺ T cells in anindividual, particularly in an HIV infected individual. It has beenshown that when CD4⁺ T cells infected with HIV are cultured in vitrowith anti-CD3 and anti-CD28 attached to a solid phase surface, expansionof the T cell population is obtained and the amount of HIV produced issignificantly reduced compared to the amount of virus produced when thecells are stimulated with PHA and IL-2 (Example 15). Thus, in oneembodiment of the invention, expansion of the population of CD4⁺ T cellsin an HIV infected individual is achieved by administration to theindividual of anti-CD3 and anti-CD28 antibodies attached to a solidphase surface. This particular embodiment should be useful as atherapeutic method for increasing the number of CD4⁺ T cells in anindividual, since the expansion of the T cells will occur with limitedreduced HIV replication.

The methods for stimulating and expanding a population of antigenspecific T cells are useful in therapeutic situations where it isdesirable to upregulate an immune response (e.g., induce a response orenhance an existing response) upon administration of the T cells to asubject. For example, the method can be used to enhance a T cellresponse against tumor-associated antigens. Tumor cells from a subjecttypically express tumor-associated antigens but may be unable tostimulate a costimulatory signal in T cells (e.g., because they lacksexpression of costimulatory molecules). Thus, tumor cells can becontacted with T cells from the subject in vitro and antigen specific Tcells expanded according to the method of the invention and the T cellsreturned to the subject. Alternatively, T cells can be stimulated andexpanded as described herein to induce or enhance responsiveness topathogenic agents, such as viruses (e.g., human immunodeficiency virus),bacteria, parasites and fungi.

The invention further provides methods to selectively expand a specificsubpopulation of T cells from a mixed population of T cells. Inparticular, the invention provides a method to specifically enrich apopulation of CD28⁺ T cells in CD4⁺ T cells. Indeed, stimulation of apopulation of CD28⁺ T cells with anti-CD3 and anti-CD28 antibodies or anatural ligand of CD28, such as B7-1 or B7-2 present on the surface ofCHO cells results in expansion of the population of CD4⁺ T cells at theexpense of the CD8⁺ T cells, which progressively die by apoptosis (seeExample 15). Thus, expansion of CD28⁺ T cells under these conditionsresults in a selective enrichment in CD4⁺ T cells in long term cultures.

Another embodiment of the invention, provides a method for selectivelyexpanding a population of either TH11 or TH2 cells or from a populationof CD4⁺ T cells. A population of CD4⁺ T cells can be enriched in eitherTH1 or TH2 cells by stimulation of the T cells with a first agent whichprovides a primary activation signal and a second agent which provides aCD28 costimulatory signal i.e., an anti-CD28 antibody or a naturalligand for CD28, such as B7-1 or B7. For example, to selectively expandTH2 cells from a population of CD4⁺ cells, CD4⁺ T cells are costimulatedwith a natural ligand of CD28, such as B7-1 or B7-2, present on thesurface of cells, such as CHO cells, to induce secretion of TH2 specificcytokines, such as IL-4 and IL-5, resulting in selective enrichment ofthe T cell population in TH2 cells. On the contrary, to expand TH1 cellsfrom a population of CD4⁺ T cells, CD4⁺ T cells are costimulated with ananti-CD28 antibody, such as the monoclonal antibody 9.3, inducingsecretion of TH1-specific cytokines, including IFN-γ, resulting inenrichment of TH1 cells over TH2 cells (Example 14).

Compositions and Kits

This invention also provides compositions and kits comprising an agentwhich stimulates an accessory molecule on the surface of T cells (e.g.,an anti-CD28 antibody) coupled to a solid phase surface and, optionally,including an agent which stimulates a TCR/CD3 complex-associated signalin T cells (e.g., an anti-CD3 antibody) coupled to the same solid phasesurface. For example, the composition can comprise an anti-CD28 antibodyand an anti-CD3 antibody coupled to the same solid phase surface (e.g.bead). Alternatively, the composition can include an agent whichstimulates an accessory molecule on the surface of T cells coupled to afirst solid phase surface and an agent which stimulates a TCR/CD3complex-associated signal in T cells coupled to a second solid phasesurface. For example, the composition can include an anti-CD28 antibodycoupled to a first bead and an anti-CD3 antibody coupled to a secondbead. Kits comprising such compositions and instructions for use arealso within the scope of this invention.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references andpublished patent applications cited throughout this application arehereby incorporated by reference. The following methodology described inthe Materials and Methods section was used throughout the examples setforth below.

METHODS AND MATERIALS

Preparation of Immobilized Anti-CD3 Antibody

Tissue culture flasks were coated with anti-CD3 monoclonal antibody.Although a number of anti-human CD3 monoclonal antibodies are available,OKT3 prepared from hybridoma cells obtained from the American TypeCulture Collection was used in this procedure. For any anti-CD3 antibodythe optimal concentration to be coated on tissue cultured flasks must bedetermined experimentally. With OKT3, the optimal concentration wasdetermined to be typically in the range of 0.1 to 10 micrograms permilliliter. To make coating solution, the antibody was suspended in 0.05M tris-HCl,pH 9.0 (Sigma Chemical Co., St. Louis, Mo.). Coating solutionsufficient to cover the bottom of a tissue culture flask was added(Falcon, Nunc or Costar) and incubated overnight at 4° C. The flaskswere washed three times with phosphate buffered saline without calciumor magnesium (PBS w/o Ca or Mg) and blocking buffer (PBS w/o Ca or Mgplus 5% bovine serum albumin) added to cover the bottom of the flask andwere incubated two hours at room temperature. After this incubation,flasks were used directly or frozen for storage, leaving the blockingsolution on the flask.

Isolation of Peripheral Blood Leukocytes (PBLs)

Samples were obtained by leukopheresis of healthy donors. Using sterileconditions, the leukocytes were transferred to a T800 culture flask. Thebag was washed with Hanks balanced salt solution w/o calcium ormagnesium (HBSS w/o) (Whittaker Bioproducts, Inc., Walkersville, Md.).The cells were diluted with HBSS w/o and mixed well. The cells were thensplit equally between two 200 milliliter conical-bottom sterile plastictissue culture tubes. Each tube was brought up to 200 ml with HBSS w/oand spun at 1800 RPM for 12 minutes in a Beckman TJ-6 centrifuge. Thesupernatant was aspirated and each pellet resuspended in 50 ml HBSS w/o.The cells were transferred to two 50 ml conical bottom tubes and spun at1500 RPM for eight minutes. Again the supernatant was aspirated.

To lyse the red blood cells, the cell pellets were resuspended in 50 mlof ACK lysing buffer (Biofluids, Inc., Rockville Md., Catalog #304) atroom temperature with gentle mixing for three minutes. The cells wereagain pelleted by spinning at 1500 RPM for 8 minutes. After aspiratingthe supernatant, the pellets were combined into one 50 ml tube in 32 mlHBSS w/o.

Separation of the PBLs from monocytes was accomplished by centrifugationthrough a PERCOLL™ gradient. To prepare 1 liter of PERCOLL™ solution(PERCOLL™-MO), 716 ml of PERCOLL™ (Pharmacia, Piscataway, N.J., Catalog#17-0891-01) was combined with 100 ml 1.5 M sodium chloride, 20 ml 1Msodium-HEPES, and 164 ml water. All reagents must be tissue culturegrade and sterile filtered. After mixing, this solution was filteredthrough a sterile 0.2 μm³ filter and stored at 4° C. 24 ml ofPERCOLL™-MO was added to each of two 50 ml conical bottom tubes. To eachtube 16 ml of the cell suspension was added. The solution was mixed wellby gently inverting the tubes. The tubes were spun at 2800 RPM for 30minutes without a brake. The tubes were removed from the centrifuge,being careful not to mix the layers. The PBLs were at the bottoms of thetubes. Then, the supernatant was aspirated and the PBLs were washed inHBSS w/o by centrifuging for 8 minutes at 1500 RPM.

Cell Sorting Via Negative Magnetic Immunoadherence

The cell sorting via negative magnetic immunoadherence must be performedat 4° C. The washed cell pellets obtained from the PERCOLL™ gradientsdescribed above were resuspended in coating medium (RPMI-1640 (BioWhittaker, Walkersville, Md., Catalog # 12-167Y), 3% fetal calf serum(FCS) (or 1% human AB serum or 0.5% bovine serum albumin) 5 mM EDTA(Quality Biological, Inc., Gaithersburg, Md., Catalog # 14-117-1), 2 mML-glutamine (Bio Whittaker, Walkersville, Md., Catalog # 17-905C), 20 mMHEPES (Bio Whittaker, Walkersville, Md., Catalog # 17-757A), 50 μg/mlgentamicin (BioWhittaker, Walkersville, Md., Catalog # 17-905C)) to acell density of 20×10⁶ per ml. A cocktail of monoclonal antibodiesdirected to cell surface markers was added to a final concentration of 1μg/ml for each antibody. The composition of this cocktail is designed toenrich for either CD4⁺ or CD28⁺ T cells. Thus, the cocktail willtypically include antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and(for CD4⁺ cells only) CD8. (See Table 1 for a list of sorting monoclonalantibody cocktails.) The tube containing cells and antibodies wasrotated at 4° C. for 30-45 minutes. At the end of this incubation, thecells were washed three times with coating medium to remove unboundantibody. Magnetic beads coated with goat anti-mouse IgG DYNABEADS®M450,Catalog #11006, P&S Biochemicals, Gaithersburg, Md.) and prewashed withcoating medium were added at a ratio of three beads per cell. The cellsand beads were then rotated for 1-1.5 hours at 4° C. The antibody-coatedcells were removed using a magnetic particle concentrator according tothe manufacturer's directions (MPC-1, Catalog # 12001, P&S Biochemicals,Gaithersburg, Md.). The nonadherent cells were washed out of the coatingmedium and resuspended in an appropriate culture medium.

TABLE 1 Sorting Monoclonal Antibody Cocktails: (Italicized mAbs areavailable from the ATCC) Cocktail Targets Representative mAbs rt-A CD1463D3 (IgG1) 20.3 (IgM) CD20 IF5 (IgG2_(a)), Leu-16 (IgG1) CD16 FC-2.2(IgG2_(b)), 3G8 (IgG1) HLA-DR 2.06 (IgG1), HB10a (IgG) rT-B CD14 63D3(IgG1), 20.3 (IgM) CD21 HB5 (IgG2_(a)) CD16 FC-2.2 (IgG2_(b)), 3G8(IgG1) HLA-DR 2.06 (IgG1), HB10a (IgG) r9.3-A CD14 63D3 (IgG1), 20.3(IgM) CD20 IF5 (IgG2_(a)), Leu-16 (IgG1) CD11b OKMI (IgG2_(b)), 60.1(IgG2_(b)) CD16 FC-2.2 (IgG2_(b)), 3G8 (IgG1) HLA-DR 2.06 (IgG1), HB10a(IgG) r9.3-B CD14 63D3 (IgG1), 20.3 (IgM) CD21 HB5 (IgG2_(a)) CD11b OKMI(IgG2_(b)), 60.1 (IgG2_(b)) CD16 FC-2.2 (IgG2_(b)), 3G8 (IgG1) HLA-DR2.06 (IgG1), HB10a (IgG) rCD4-A CD14 63D3 (IgG1), 20.3 (IGM) CD20 IF5(IgG2_(a)), Leu-16 (IGg1) CD11b OKMI (IgG2_(b)), 60.1 (IgG2_(b)) CD16FC-2.2 (IgG_(b)), 3G8 (IgG1) HLA-DR 2.06 (IgG1), HB10a (IgG) CD8 51.1(IgG2), G10-1.1 (IgG2_(a)), OKT8, (IgG2_(a)) rCD8-B CD14 63D3 (IgG1),20.3 (IgM) CD20 IF5 (IgG2_(a)), Leu-16 (IGg1) CD11b OKMI (IgG2_(b)),60.1 (IgG2_(b)) CD16 FC-2.2 (IgG2_(b)), 3G8 (IgG1) HLA-DR 2.06 (IgG1),HB10a (IgG) CD4 G17-2.8 (IgG1) rM0 CD2 35.1 (IgG2_(a)), 9.6 (IgG2_(a))CD20 IF5 (IgG2_(a)), Leu-16 (IGg1) rB CD2 35.1 (IgG2_(a)), 9.6(IgG2_(a)) CD14 63D3 (IgG1), 20.3 (IgM) CD11b OKMI (IgG2_(b), 60.1(IgG2_(b)) CD16 FC-2.2 (IgG2_(b)), 3G8 (IgG1)Long Term Stimulation:

Tissue culture flasks precoated with anti-CD3 monoclonal antibody werethawed and washed three times with PBS. The purified T cells were addedat a density of 2×10⁶/ml. Anti-CD28 monoclonal antibody mAb 9.3 (Dr.Jeffery Ledbetter, Bristol Myers Squibb Corporation, Seattle, Wash.) orEX5.3D10, ATCC Deposit No. HB11373 (Repligen Corporation, Cambridge,Mass.) was added at a concentration of 1 μg/ml and cells were culturedat 37° C. overnight. The cells were then detached from the flask byforceful pipetting and transferred to a fresh untreated flask at adensity of 0.5×10⁶/ml. Thereafter, the cells were resuspended everyother day by forceful pipetting and diluted to 0.5×10⁶/ml. The meandiameter of the cells was monitored daily with a COULTER COUNTER®2Minterfaced to a Coulter Channelyzer. Resting T cells have a meandiameter of 6.8 microns. With this stimulation protocol, the meandiameter increased to over 12 microns by day 4 and then began todecrease by about day 6. When the mean diameter decreased to about 8microns, the cells were again stimulated overnight with anti-CD3 andanti-CD28 as above. It was important that the cells not be allowed toreturn to resting diameter. This cycle was repeated for as long as threemonths. It can be expected that the time between restimulations willprogressively decrease.

Example 1

Long Term Growth of CD4⁺ T cells With Anti-CD3 and Anti-CD28 Antibodies

Previous known methods to culture T cells in vitro require the additionof exogenous feeder cells or cellular growth factors (such asinterleukin 2 or 4) and a source of antigen or mitogenic plant lectin.Peripheral blood CD28⁺ T cells were isolated by negative selection usingmagnetic immunobeads and monoclonal antibodies as described in theMethods and Materials section above. CD4⁺ cells were further isolatedfrom the T cell population by treating the cells with anti-CD8monoclonal antibody and removing the CD8⁺ cells with magneticimmunobeads. Briefly, T cells were obtained from leukopheresis of anormal donor, and purified with FICOLL™ density gradient centrifugation,followed by magnetic immunobead sorting. The resulting CD28⁺, CD4⁺ Tcells were cultured in defined medium (X-Vivo10 containing gentamicinand L-glutamine (Whittaker Bioproducts) at an initial density of2.0×10⁶/ml by adding cells to culture dishes containing plastic-adsorbedGoat anti-mouse IgG (Kirkegaard and Perry Laboratories, Gaithersburg,Md.) and anti-CD3 mAb G19-4. After 48 hours, the cells were removed andplaced in flasks containing either hIL-2 (5%, CalBiochem) or anti-CD28mAb (500 ng/ml). The cells cultured with IL-2 were fed with fresh IL-2at 2-day intervals. Fresh medium was added to all cultures as requiredto maintain a cell density of 0.5×10⁶/ml. Cells were restimulated atapproximately weekly intervals by culture on plastic-adsorbed anti-CD3mAb for 24 hours, the cells removed and placed at 1.0×10⁶/ml in freshmedium in flasks containing either IL-2 or anti-CD28 mAb.

In the example shown in FIG. 1, the culture vessel initially contained50×10⁶ cells, and the cells were cultured in an optimal amount ofmitogenic lectin PHA, or cultured with cyclic stimulation of plasticimmobilized anti-CD3 mAb in the presence of interleukin 2 or anti-CD28mAb 9.3. The cells cultured in PHA alone did not proliferate, with allcells dying by about day 20 of culture, demonstrating the functionalabsence of accessory cells. In contrast, the cells grown in anti-CD3with IL-2 or anti-CD28 entered a logarithmic growth phase, with equalrates of growth for the first three weeks of culture. However, theanti-CD3 cultures began to diverge in growth rates during the fourthweek of culture, with the IL-2 fed cells entering a plateau phase aftera ^(˜)2.8log₁₀ expansion. In contrast, the cultures grown in thepresence of anti-CD28 remained in logarithmic growth until the sixthweek of culture, at which time there had been a ^(˜)3.8log₁₀ expansion.Thus, CD28 receptor stimulation, perhaps by anti-CD28 crosslinking, isable to stimulate the growth of CD4⁺ T cells in the absence of fetalcalf serum or accessory cells, and furthermore, about 10-fold more cellscan be obtained using anti-CD28 as opposed to addition of exogenousIL-2. In repeated experiments, CD4⁺ T cell expansion using anti-CD28antibody consistently yielded more CD4⁺ T cells than expansion usingIL-2 (e.g., up to 1000-fold more cells). This system has the addedadvantage of not requiring the presence of accessory cells which may beadvantageous in clinical situations where accessory cells are limitingor defective.

Example 2

Long Term Growth of Anti-CD28-Treated T cells In Medium Containing FetalCalf Serum

Another series of experiments tested whether the growth advantage ofCD28 receptor stimulation was due to replacement of factors normallypresent in fetal calf serum. T cells were obtained from leukopheresis ofa normal donor, and purified with FICOLL™ density gradientcentrifugations, followed by magnetic immunobead sorting. The resultingCD28⁺ , CD4⁺ T cells were cultured at an initial density of 2.0×10⁶/mlin medium (RPMI-1640 containing 10% heat-inactivated fetal calf serum[Hyclone, Logan, Utah] and gentamicin and L-glutamine) by adding cellsto culture dishes containing plastic-adsorbed OKT3. After 48 hours, thecells were removed and placed in flasks containing either hIL-2 (10%final concentration, CalBiochem) or anti-CD28 mAb 9.3 (800 ng/ml). Thecells were fed with fresh medium as required to maintain a cell densityof 0.5×10⁶/ml, and restimulated at approximately weekly intervals byculture on plastic adsorbed anti-CD3 mAb for 24 hours.

As shown in FIG. 2, the cells entered logarithmic growth phase, withequal rates of growth for the first three weeks of culture. However, theanti-CD3 cultures began to diverge in growth rates during the fourthweek of culture, with the IL-2 fed cells entering a plateau phase aftera ^(˜)4.1log₁₀ expansion. In contrast, the cultures grown in thepresence of anti-CD28 remained in logarithmic growth until the fifthweek of culture, at which time there had been a ˜5.1log₁₀ expansion.Thus, CD28 stimulation resulted in a ^(˜)125,000-fold expansion of theinitial culture while IL-2 feeding resulted in a 10,000-fold expansionof cells.

Example 3

Long Term Growth of T cells in Phorbol Ester. Ionomycin andAnti-CD28-Stimulated T cells

Further experiments tested whether alternative methods of activating Tcells would also permit CD28 stimulated growth. Pharmacologic activationof T cells with PMA and ionomycin is thought to mimic antigen receptortriggering of T cells via the TCR/CD3 complex. T cells were obtainedfrom leukopheresis of a normal donor, and purified with sequentialFICOLL™ and PERCOLL™ density gradient centrifugations, followed bymagnetic immunobead sorting. The resulting CD28⁺, CD4⁺ T cells werecultured at an initial density of 2.0×10⁶/ml by adding cells to culturedishes containing phorbol myristic acid (PMA 3 ng/ml, Sigma) andionomycin (120 ng/ml, Calbiochem, lot #3710232). After 24 hours, thecells were diluted to 0.5×10⁶/ml and placed in flasks containing eitherrIL-2 (50 IU/ml, Boerhinger Mannheim, lot #11844900)) or anti-CD28 mAb(1 ug/ml). The cells were fed with fresh medium as required to maintaina cell density of 0.5×10⁶/ml, and restimulated cyclically atapproximately weekly intervals by readdition of PMA and ionomycin. FreshIL-2 was added to the IL-2 containing culture at daily intervals.

The results of this experiment are shown in FIG. 3. T cells that werepurified of accessory cells did not grow in cell numbers in the presenceof PMA (“P” in the Figure) and ionomycin (“I” in the Figure), with orwithout IL-2. The cells clumped and enlarged, as indicated by sizeanalysis, indicating the cells had been induced to enter the G1 phase ofthe cell cycle but did not progress to DNA synthesis and cell division.In contrast, addition of CD28 mAb to PMA plus ionomycin treated cellsresulted in logarithmic cell growth. Thus, anti-CD3 mAb is not requiredto provide T cell activation. It should be appreciated that otheractivators of protein kinase C, such as bryostatin or diacylglycerol canbe used in place of PMA.

Example 4

Immunophenotype of Cells Cultured with Anti-CD3 Stimulation and Additionof IL-2 or Anti-CD28 mAb

To examine the subsets of T cells that are expanded, PBL were propagatedfor 16 days using either anti-CD3 and IL-2 or anti-CD3 and anti-CD28.FIG. 4 demonstrates the selective enrichment of CD4 cells fromperipheral blood lymphocytes. Mononuclear cells were isolated from bloodby ficoll hypaque density gradient centrifugation. The cells werestained with CD4 and CD8 monoclonal antibodies, and analyzed for thepercent positive cells on day 0. The cells were then cultured on plasticimmobilized anti-CD3 monoclonal antibody G 19-4 plus IL-2 or plasticimmobilized anti-CD3 monoclonal antibody G19-4 plus anti-CD28 monoclonalantibody 9.3 (0.5 μg/ml). The cells were isolated from culture on day16, and repeat staining for CD4 and CD8 antigens was done by flowcytometry. Data was gated on the lymphocyte population by forward anglelight scatter and side scatter. By this analysis, the % CD4 and CD8cells were 8.0% and 84.5% in the cells grown in IL-2, and 44.6% and52.5% in the cells grown in CD28. These results suggest that CD28expansion favors the CD4⁺ cell, in contrast to the well-establishedobservation that CD8⁺ cells predominate in cells grown in IL-2 (forexample, see D. A. Cantrell and K. A. Smith, (1983), J. Exp. Med.158:1895 and Gullberg, M. and K. A. Smith (1986) J. Exp. Med. 163, 270).

To further test this possibility, CD4⁺ T cells were enriched to 98%purity using negative selection with monoclonal antibodies and magneticimmunobeads as described above. Fluorescent Activated Cell Sorter (FACS)Analysis was used to examine the phenotype of the T cells cultured withanti-CD3 and anti-CD28. Cells were pelleted by centrifugation andresuspended in PBS/1% BSA. The cells were then washed by repeating thisprocedure twice. The cells were pelleted and resuspended in 100 μl ofprimary antibody solution, vortexed, and kept on ice for one hour. Afterwashing twice in PBS/1% BSA, the cells were resuspended in 100 μl offluorescein-labeled goat-anti-mouse IgG and incubated for 30 minutes onice. At the end of this incubation, the cells were washed twice in PBSand resuspended in 500 μl 1% paraformaldehyde in PBS. The labeled cellswere analyzed on an Ortho Cytofluorograph. Cells were stained afterisolation, or after 26 days in culture, with phycoerythrin conjugatedanti-CD3 (Leu-4), CD4 (Leu-3A), CD8 (OKT8) or with IgG2a controlmonoclonal antibodies and fluorescence quantified with a flow cytometer.The cells were cultured for one month using anti-CD3 and either IL-2 oranti-CD28 to propagate the cells. There was equal expansion of the cellsfor the first 26 days of the culture (not shown), however, as can beseen in FIG. 5, the phenotype of the cells diverged progressively withincreasing time in culture so that at day 26 of culture, the predominantcell in anti-CD28 culture was CD4⁺ while the cells in the IL-2 culturewere predominantly CD8⁺. Thus, CD28 receptor stimulation, perhaps bycrosslinking, is able to selectively expand T cells of the CD4 phenotypewhile the conventional method of in vitro T cell culture yields cells ofthe CD8 phenotype. Additional experiments have been conducted withsimilar results, indicating that CD28 stimulation of initially mixedpopulations of cells is able to yield cultures containing predominatelyor exclusively CD4 T cells, and thus one can expand and “rescue” the CD4cells that were initially present in limiting amounts.

Example 5

Use of Cell Sizing or Cyclic Expression of B7 on CD4⁺ T cells to MonitorT Cell Expansion

To determine the time of T cell restimulation, changes in cell volumewere monitored using a COULTER COUNTER®ZM interfaced with a Coulter.CD28⁺CD4⁺ T cells were isolated as described by magneticimmunoselection, and cultured in the presence of anti-CD28 mAb 9.3 (0.5μg/ml) and restimulated with plastic immobilized anti-CD3 monoclonalantibody G19-4 as indicted. FIG. 9 demonstrates the cyclic changes incell volume during six consecutive restimulations (“S1” to “S6”)performed essentially as described in Example 1. Briefly, cells wereexpanded with anti-CD3 and anti-CD28 over three weeks in culture. Cellswere changed to fresh medium at each restimulation with anti-CD3antibody. Stimulations were spaced at ten day intervals. The cells wererestimulated whenever cell volume decreased to <400 fl.

In another experiment, cyclic expression of the B7-1 antigen was used todetermine the time for T cell restimulation. The cells obtained from theexperiment shown in FIG. 10 were stained with a CTLA-4Ig fusion protein(obtained from Repligen Corporation; see also Linsley P. S. et al.(1991) J. Exp. Med. 174, 561-569) and analyzed by flow cytometry tomeasure B7-1 receptor expression. It was determined that CD4⁺ T cells donot initially express the B7-1 receptor, and that with culture,expression is induced. Further, the B7-1 expression was found to betransient, and to be re-induced with repeated anti-CD3 restimulation.

Example 6

Production of Cytokines by T Cells Following Anti-CD28 Stimulation

Experiments were conducted to analyze the cytokines produced by T cellsfollowing anti-CD28 stimulation. CD28⁺/CD4⁺ T cells were isolated asdescribed in the previous examples. The cells were stimulated withplastic immobilized anti-CD3 mAb and IL-2 (200 U/ml), or anti-CD3 andanti-CD28 without added lymphokine. The cells were restimulated withanti-CD3 antibody as determined by changes in cell volume as describedin Example 5. Cell culture supernatant was removed at the time pointsindicated and analyzed for IL-2 (FIG. 11), GM-CSF (FIG. 12), and TNF-a(FIG. 13). IL-2 was determined by bioassay on CTLL-2 cells while TNFαand GM-CSF were measured by ELISA according to manufacturersinstructions (TNFα, GMCSF:R&D Systems, Minneapolis, Minn.). The datashown for the various cytokines are from separate experiments. In otherexperiments (not shown) anti-CD3 plus anti-CD28 stimulation was shown tocause high levels of IL4 and IL-5 in culture supernatants afterapproximately day 10 of culture, although only small amounts of thesecytokines were present during the early period of culture.

The patterns of cytokine secretion with cells expanded by severalrestimulations according to the protocol described in the examples wascompared to cells expanded with anti-CD3 plus IL-2 over three weeks inculture. Cells were changed to fresh medium at each restimulation withanti-CD3 antibody. Stimulations were spaced at ten day intervals. After24 hours of further culture, an aliquot of cell culture supernatant wasremoved for assay. ELISA assays for individual cytokines were performedwith kits from various suppliers (IL-2:T Cell Diagnostics, Cambridge,Mass.; IFN-γ Endogen, Inc., Boston, Mass.; IL-4, TNFα, GMCSF:R&DSystems, Minneapolis, Minn.) according to directions supplied with thekits. As can be seen from the results of a representative experimentshown in Table 2, the two protocols result in very similar levels ofIL-2 and IL-4 secretion. The higher levels of GM-CSF and TNFα secretionwith anti-CD3 and anti-CD28 costimulation suggests that theproliferative capacity of this combination of stimuli may be due in partto its ability to stimulate an autocrine loop.

TABLE 2 Comparison of cytokines secreted by T cells expanded withanti-CD3 and IL-2 versus T cells expanded with anti-CD3 and anti-CD28.Stimulation Costimu- Concentration of lymphokine in pg/ml cycle lus IL-2IFN-γ IL-4 GM-CSF TNFα S1 IL-2 20714 1458 16 2303 789 αCD28 13794 221114 3812 3387 S2 IL-2 20250 16600 964 51251 3221 αCD28 28411 56600 1030138207 13448 S3 IL-2 21282 8617 1153 86418 2899 αCD28 14129 12583 1044120418 5969

Example 7

Polyclonality of T Cells Following Anti-CD28 Stimulation

The polyclonality of a population of T cells following stimulation withan anti-CD3 and an anti-CD28 antibody as described in the precedingexamples was determined. CD28⁺/CD4⁺ T cells were isolated as describedin the previous examples. The cells were stimulated with plasticimmobilized anti-CD3 mAb and anti-CD28 mAb and FACS analysis conductedessentially as described in Example 4 using a panel of anti-TCRantibodies (Vpβ5a, Vβ5b, Vβ5c, Vβ6a, Vβ8a, Vβ12a and Vα2a) obtained fromPharmingen. The polyclonality of the T cell population was determinedbefore (Day 1) and after stimulation (Day 24). As shown in FIG. 14, theTCR diversity of a population of T cells stimulated through CD28 ismaintained at day 24.

Example 8

Comparison of Cell Surface Staining of T Cells from HIV⁺ andHIV-Individuals Following Anti-CD28 Stimulation

Another series of experiments was conducted to determine the expressionof various T cell surface markers on cells from HIV seropositive andseronegative individuals expanded according to the procedures describedin the previous examples. CD28⁺/CD4⁺ T cells were obtained as describedherein. In these experiments, the anti-CD3 mAb was labeled with a firstlabel (e.g., rhodamine) and the appropriate second antibody (e.g.,anti-CD28, anti-CD4, anti-CD8) was labeled with a second label (e.g.,fluorescein). T cells were stimulated with plastic immobilized anti-CD3mAb and anti-CD28 in mAb as described herein and the percent of T cellsexpressing a variety of cell surface markers at different stimulations(i.e., S1, S2 and S3) determined by FACS analysis. As shown in FIGS. 15and 16, the overall cell surface marker distribution on T cells obtainedfrom HIV seropositive and seronegative individuals is approximately thesame throughout the stimulation assay. It is noteworthy that thepresence of one cell surface marker, CD45RA, which is a marker for naiveT cells, declines over the course of CD28 stimulated T cell expansion.In contrast, the percent of T cells expressing the memory T cell surfacemarker, CD45RO, increases with CD28 stimulation. Thus, T cell expansionthrough CD28 stimulation preferentially expands memory T cells orconverts naive T cells to memory T cells. It should be noted that thedecline in the percent of T cells expressing CD28 is an artifact of theexperiment due to the presence of anti-CD28 antibody in the T cellculture throughout the assay. The presence of anti-CD28 antibodyprevents staining of the CD28 antigen.

Example 9

Long Term Growth of CD8⁺ T cells With Anti-CD3 and Monoclonal AntibodyES5.2D8

Experiments were conducted to determine whether a population of CD8⁺ Tcells could be preferentially expanded by stimulation with an anti-CD3mAb and a monodonal antibody ES5.2D8. CD28⁺ T cells were obtainedessentially as described in Example 1. To assay for CD8 expression, aprimary anti-CD8 antibody and a labeled appropriate secondary antibodywere used in FACS analysis to determine the percent positive cells. Asshown in FIG. 17, at day 7 following stimulation of T cells with theanti-CD3 mAb G19-4sp and the mAb ES5.2D8 the CD8⁺ fraction had increasedfrom approximately 20% to over 40%. Another monoclonal antibody ER4.7G11(referred to as 7G11) was also found to stimulate CD8⁺ T cells. Thisantibody was raised against recombinant human CTLA4 and has beendeposited with the ATCC on Jun. 3, 1994 at Accession No. HB 11642. Thisresult indicates that binding of either a distinct region of CTLA4 or ofa cross-reactive cell surface protein selectively activates CD8⁺ Tcells.

Example 10

Defining the Epitope of the Monoclonal Antibody ES5.2D8 and Cloning theCD9 Antigen

To determine the epitope of the monoclonal antibody ES5.2D8 epitopemapping was performed by phage display library (PDL) screening and wasconfirmed using synthetic peptides. A random 20 amino acid PDL wasprepared by cloning a degenerate oligonucleotide into the fUSE5 vector(Scott, J. K. and Smith, G. P. (1990) Science 249:386-390) as describedin Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382.The PDL was used to identify short peptides that specifically bound mAbES5.2D8 by a micropanning technique described in Jellis, C. L. et al.(1993) Gene 137:6368. Individual phage clones were purified from thelibrary by virtue of their affinity for immobilized mAb and the randompeptide was identified by DNA sequencing. Briefly, mAb ES5.2D8 wascoated onto Nunc Maxisorp 96 well plates and incubated with 5×10¹⁰ phagerepresenting 8×10⁶ different phage displaying random 20 amino acidpeptides. Specifically bound phage were eluted, amplified, thenincubated with the antibody a second time. After the third round, 7phage were isolated, and DNA was prepared for sequencing.

Sequence analysis of these clones demonstrated that three of the sevensequences were identical and a fourth was similar:

-   -   2D8#2(SEQ ID NO:8) H Q F C D H W G C W L L R E T H I F T P    -   2D8#4(SEQ ID NO:8) H Q F C D H W G C W L L R E T H I F T P    -   2D8#10(SEQ ID NO:8) H Q F C D H W G C W L L R E T H I F T P    -   2D8#6(SEQ ID NO:9) L R L V L E D P G I W L R P D Y F F P A        Based on this data an epitope of G X W L X D/E (SEQ ID NO: 12)        was proposed.

In addition to CITLA4, a second antigen for mAb ES5.2D8 was discoveredusing cDNA expression cloning.

A. Construction oa cDNA Expression Library

A cDNA library was constructed in the pCDM8 vector (Seed, (1987) Nature32:840) using poly (A)⁺ RNA isolated from activated T cells as described(Aruffo et al. (1987) Proc. Natl. Acad. Sci. USA 84:3365). To preparetotal RNA, T cells were harvested from culture and the cell pellethomogenized in a solution of 4 M guanidine thiocyanate, 0.5% sarkosyl,25 mM EDTA, pH 7.5, 0.13% Sigma anti-foam A, and 0.7% mercaptoethanol.RNA was purified from the homogenate by centrifugation for 24 hour at32,000 rpm through a solution of 5.7 M CsCl, 10 mM EDTA, 25 mM Naacetate, pH 7. The pellet of RNA was dissolved in 5% sarkosyl, 1 mMEDTA, 10 mM Tris, pH 7.5 and extracted with two volumes of 50% phenol,49% chloroform, 1% isoamyl alcohol. RNA was ethanol precipitated twice.Poly (A)⁺ RNA used in cDNA library construction was purified by twocycles of oligo (dT)-cellulose selection.

Complementary DNA was synthesized from 5.5 μg of poly(A)⁺ RNA in areaction containing 50 mM Tris, pH 8.3, 75 mM KCl, 3 mM MgCl₂, 10 mMdithiothreitol, 500 μM dATP, dCTP, dGTP, dTTP, 50 μg/ml oligo(dT)₁₂₋₁₈,180 units/ml RNasin, and 10,000 units/ml Moloney-MLV reversetranscriptase in a total volume of 55 μl at 37° C. for 1 hr. Followingreverse transcription, the cDNA was converted to double-stranded DNA byadjusting the solution to 25 mM Tris, pH 8.3, 100 mM KCl, 5 mM MgCl₂,250;M each dATP, dCTP, dGTP, dTTP, 5 mM dithiothreitol, 250 units/ml DNApolymerase 1, 8.5 units/ml ribonuclease H and incubating at 16° C. for 2hr. EDTA was added to 18 mM and the solution was extracted with an equalvolume of 50% phenol, 49% chloroform, 1% isoamyl alcohol. DNA wasprecipitated with two volumes of ethanol in the presence of 2.5 Mammonium acetate and with 4 micrograms of linear polyacrylamide ascarrier. In addition, cDNA was synthesized from 4 μg of poly(A)⁺ RNA ina reaction containing 50 mM Tris, pH 8.8, 50 μg/ml oligo(dT)₁₂₋₁₈, 327units/ml RNasin, and 952 units/ml AMV reverse transcriptase in a totalvolume of 100 μl at 42° C. for 0.67 hr. Following reverse transcription,the reverse transcriptase was inactivated by heating at 70° C. for 10min. The cDNA was converted to double-stranded DNA by adding 320 μl H₂Oand 80 μl of a solution of 0.1M Tris, pH 7.5, 25 mM MgCl₂, 0.5 M KCl,250 μg/ml bovine serum albumin, and 50 mM dithiothreitol, and adjustingthe solution to 200 μM each dATP, dCTP, dGTP, dTTP, 50 units/ml DNApolymerase I, 8 units/ml ribonuclease H and incubating at 16° C. for 2hours. EDTA was added to 18 mM and the solution was extracted with anequal volume of 50% phenol, 49% chloroform, 1% isoarnyl alcohol. DNA wasprecipitated with two volumes of ethanol in the presence of 2.5 Mammonium acetate and with 4 micrograms of linear polyacrylamide ascarrier.

The DNA from 4 μg of AMV reverse transcription and 2.0 μg of Moloney MLVreverse transcription were combined. Non-selfcomplementary BstXIadaptors were added to the DNA as follows: The double-stranded cDNA from6 μg of poly(A)+RNA was incubated with 3.6 μg of a kinasedoligonucleotide of the sequence CTTTAGAGCACA (SEQ ID NO: 13) and 2.4 μgof a kinased oligonucleotide of the sequence CTCTAAAG (SEQ ID NO: 14) ina solution containing 6 mM Tris, pH 7.5,6 mM MgCl₂, 5mM NaCl, 350 μg/mlbovine serum albumin, 7 mM mercaptoethanol, 0.1 mM ATP, 2 mMdithiothreitol, 1 mM spermidine, and 600 units T4 DNA ligase in a totalvolume of 0.45 ml at 15° C. for 16 hours. EDTA was added to 34 mM andthe solution was extracted with an equal volume of 50% phenol, 49%chloroform, 1% isoamyl alcohol. DNA was precipitated with two volumes ofethanol in th presence of 2.5 M ammonium acetate.

DNA larger than 600 bp was selected as follows: The adaptored DNA wasredissolved in 10 mM Tris, pH 8, 1 mM EDTA, 600 mM NaCl, 0.1% sarkosyland chromatographed on a Sepharose CL-4B column in the same buffer. DNAin the void volume of the column (containing DNA greater than 600 bp)was pooled and ethanol precipitated.

The pCDM8 vector was prepared for cDNA cloning by digestion with BstXIand purification on an agarose gel. Adaptored cDNA from 6 μg of poly(A)⁺RNA was ligated to 2.25 μg of BstXl cut pCDM8 in a solution containing 6mM Tris, pH 7.5, 6 mM MgCl₂, 5 mM NaCl, 350 μg/ml bovine serum albumin,7 mM mercaptoethanol, 0.1 mM ATP, 2 mM dithiothreitol, 1 mM spermidine,and 600 units T4 DNA ligase in a total volume of 1.5 ml at 15° C. for 24hr. The ligation reaction mixture was then transformed into competent E.coli DH10B/P3 by standard techniques.

Plasmid DNA was prepared from a 500 ml culture of the originaltransformation of the cDNA library. Plasmid DNA was purified by thealkaline lysis procedure followed by twice banding in CsCl equilibriumgradients (Maniatis et al, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor, N.Y. (1987)).

B. Cloning Procedure

In the cloning procedure, the cDNA expression library was introducedinto MOP8 cells (ATCC No. CRL1709) using lipofectamine and the cellsscreened with mAb ES5.2D8 to identify transfectants expressing a ES5.2D8ligand on their surface. In the first round of screening, thirty 100 mmdishes of 50% confluent COS cells were transfected with 0.05 μg/mlactivated T cell library DNA using the DEAE-Dextran method (Seed, B. etal. (1987) Proc. Natl. Acad. Sci. USA 84:3365). The cells weretrypsinized and re-plated after 24 hours. After 47 hours, the cells weredetached by incubation in PBS/0.5 mM EDTA, pH 7.4/0.02% Na azide at 37°C. for 30 min.

Detached cells were treated with 10 μg/ml mAb ES5.2D8. Cells wereincubated with the monodonal antibody for 45 minutes at 4° C. Cells werewashed and distributed into panning dishes coated with affinity-purifiedgoat anti-mouse IgG antibody and allowed to attach at room temperature.After 3 hours, the plates were gently washed twice with PBS/0.5 mM EDTA,pH 7.4/0.02% Na azide, 5% FCS and once with 0.15M NaCl, 0.01M Hepes, pH7.4,5% FCS. Unbound cells were thus removed and episomal DNA wasrecovered from the adherent panned cells by conventional techniques.

Episomal DNA was transformed into E. coli DH10B/P3. The plasmid DNA wasre-introduced into MOP8 cells using lipofectamine and the cycle ofexpression and panning was repeated twice. Cells expressing a ES5.2D8ligand were selected by panning on dishes coated with goat anti-mouseIgG antibody. After the third round of screening, plasmid DNA wasprepared from individual colonies and transfected into MOP8 cells by theDEAE-Dextran method. Expression of a ES5.2D8 ligand on transfected MOP8cells was analyzed by indirect immunofluorescence with mAb ES5.2D8 (SeeFIG. 18).

DNA from one clone (mp5) identified as positive by FACS analysis wassequenced using standard techniques. FASTA analysis of the amino acidsequence of mp5 identified a matching protein, CD9, in the GCG databanks. The full amino acid sequence of CD9 is shown below (SEQ ID NO:6).

BESTFIT analysis of the phage epitopes of mAb ES5.2D8 to the amino acidsequence of CD9 revealed a dose match:

-   -   G C W L L R E (phage 2D8#2,4, 10; SEQ ID NO: 11)    -   G I W L R P D (phage 2D8#6; SEQ ID NO: 12)    -   G L W L R F D(CD9sequence; SEQ ID NO: 13)

FT DOMAIN 111 194 EXTRACELLULAR (PROBABLE) FT TRANSMEM 195 220 POTENTIALFT DOMAIN 221 227 CYTOPLASMIC (PROBABLE) FT CARBOHYD  51 51 POTENTIAL FTCARBOHYD  52 52 POTENTIAL FT CONFLICT  8 8 C → S (IN REF. 1) FT CONFLICT 66 66 G → A (IN REF. 1) FT CONFLICT 193 193 MISSING (IN REF. 1) SQSEQUENCE 227 AA; 25285 MW; 261251 CN;Cd9_Human Length: 227 May 25, 1994 14:10 Type: P Check: 1577(SEQ ID NO: 6)

-   -   1 PVKGGTKCIK YLLFGFNFIF WLAGIAVLAI GLWLRFDSQT KSIFEQETNN    -   51 NNSSFYTGVY ILIGAGALMM LVGFLGCCGA VQESQCMLGL FFGFLLVIFA    -   101 IEIAAAIWGY SHKDEVIKEV QEFYKDTYNK LKTKDEPQRE TLKAIHYALN    -   151 CCGLAGGVEQ FISDICPKKD VLETFTVKSC PDAIKEVFDN KFHIIGAVGI    -   201 GIAVVMIFGM IFSMILCCAI RRNREMV

Example 11

Induction of T Cell Expansion by Costimulation with B7-1 or B7-2

In order to determine whether costimulation through CD28 can be providedby B7-1 and B7-2 molecules expressed on cells transfected with a nucleicacid encoding either of these molecules, Chinese Hamster Ovary (CHO)cells were transfected with human B7-1 (CD80) or B7-2 (CD 86) (Freedman,A. S. et al. (1987) J. Immunol. 137:3260-3267; Freeman, G. J. et al.(1989) J. Immunol. 143:2714-2722; Freeman, G. J. et al. (1991) J. Exp.Med. 174:625-631; Freeman, G. J. et al. (1993) Science 22:909-911;Azuma, M. et al. (1993) Nature 336:76-79; Freeman, G. J. et al. (1993)J. Exp. Med. 178:2185-2192). The cells were maintained in G418 asdescribed in Gimmi et al.(1991) PNAS 88:6575 and Engel et al. (1994)Blood 84:1402. Briefly, a cDNA fragment of B7-1 containing nucleotides86-1213 (comprising the coding region) was inserted into the eukaryoticexpression vector pLEN (Metabolic Biosystem, Mountain View, Calif.)containing the human metallothionein IIA promoter, the simian virus 40enhancer, and the human growth hormone 3′ untranslanted region andpolyadenylation site. A cDNA fragment of B7-2 containing the codingregion was inserted into pLEN. Fifty micrograms of Pvu I linearizedB7-pLEN construct was cotransfected with 5 micrograms of linearizedSV2-Neo-SP65 into CHO cells by electroporation using the BRLelectroporator at settings of 250 V and 1600 μF. Transfectants wereselected by growth in medium containing the neomycin analogue G418sulfate (400 μg/ml) and were cloned. Mock transfected CHO cells weremade by transfection of linearized SV2-Neo-Sp65 alone.

To determine the relative expression of human B7-1 and B7-2 on thestably transfected CHO cells, the cells were stained with CTLA4Ig(obtained from Repligen Corporation) and FITC conjugated goat anti-humanIgG Fc, or with anti-B7-1 monoclonal antibody 133 (Freeman et al. J.Immunol. 137:3260 (1987)) and FITC goat anti-mouse IgM, or with anti-B70(B7-2) monoclonal antibody IT2 (obtained from Pharmingen Corporation)and FITC goat anti-mouse IgG, fixed in paraformaldehyde and fluorescenceanalyzed by flow cytometry. The B7-1-CHO cells expressed about twice asmany binding sites than the B7-2-CHO cells for CTLA4Ig. Control CHOcells (CHO-neo) consisted of CHO cells transfected with the neomycinresistance vector, and did not stain specifically for CTLA4Ig.Specificity of ligand expression was confirmed by measurement withanti-CD80 mAb BBI (Yokochi, T. et al. (1982) J. Immunol. 128:823-827)and anti-CD86 mAb IT2. Mitomycin C treatment did not affect B7expression.

In order to explore whether costimulation by the CD28 and CTLA4 ligandsB7-1 and B7-2 is similar, CD28⁺ T cells were cultured with anti-CD3 inthe presence of B7-1 or B7-2-transfected CHO cells, or control CHO-neocells (FIG. 19). For this experiment, CD28⁺ T were isolated fromperipheral blood lymphocytes by Percoll gradient centrifugation fromleukopacks obtained by apheresis of healthy donors and purified bynegative selection according to June et al. (1987) Mol. Cell Diol.7:4472. Purified T cells were cultured in RPMI 1640 containing 10%heat-inactivated fetal calf serum (Hyclone, Logan UT), 2 mM L-glutamine,and 20 mM Hepes in 96 well flat bottom microtiter plates at 37° C. in 5%CO₂. The CD28⁺ T cells (5×10⁴ cells/well) were stimulated with anti-CD3monoclonal antibody coated beads (3 beads per T cell) in the presence ofmitomycin C-inactivated CHO cells expressing B7-1, B7-2, or neomycineresistance gene only (2×10⁴ cells/well). Cells were also stimulated withanti-CD3 monoclonal antibody OKT3 coated beads in the presence ofanti-CD28 mAb 9.3 at 1 μg/ml. In preliminary experiments, the anti-CD28mAb was titered to determine the optimal amounts for induction of IL-2secretion. The anti-CD3 monoclonal antibody OKT3 (IgG2a), obtained fromthe ATCC, was bound to magnetic beads (M-450, Dynal Corp.) that werecoated with goat anti-mouse IgG by adding 150 femtograms of antibody perbead, and the beads washed extensively. In all experiments, antigenpresenting cells were first removed from the CD28⁺ T cells byimmunomagnetic bead depletion. CHO cells were inactivated bypretreatment with mitomycin C at 25 μg/ml for one hour. On days 2 to 4of culture, thymidine incorporation experiments were performed bypulsing the cultures overnight with methyl-³H-thymidine (New EnglandNeclear) with 37 kBq/well and incorporation determined by liquidscintillation spectroscopy. Results were expressed as the mean±SEM cpmof triplicate determinations.

The results of the experiment are shown graphically in FIG. 19. On day 3of culture, low level proliferation was observed in cells stimulatedwith anti-CD3 in the presence of control CHO cells, and levels ofthymidine incorporation did not increase with further culture. Incontrast, there was increasing thymidine incorporation in T cells thatwere co-cultured with CHO cells that express B7-1 or B7-2 that wasdetected after 2 to 4 days of culture, and this cellular proliferationcontinued until exhaustion of culture medium. Levels of thymidineincorporation in B7-1 and B7-2 stimulated cultures was similar tocultures stimulated with anti-CD3 plus anti-CD28 monoclonal antibody.Thus, B7-1 and B7-2 molecules expressed on CHO cells can costimulate Tcells as efficiently as anti-CD28 monoclonal antibody.

In preliminary experiments, culture of T cells with B7-1 or B7-2expressing CHO cells alone or in combination did not result in T cellproliferation, consistent with previous reports that B7-1 alone is notmitogenic (Liu et al. (1992) Eur. J. Immunol. 22:2855). Anti-CD28stimulation in combination with PMA has previously been shown to becomitogenic. To determine whether B7-1 or B7-2 were comitogenic withPMA, purified CD28⁺ T cells were cultured in PMA (10 nM) with mitomycinC-inactivated CHO cells expressing B7-1, B7-2, a 1:1 mixture of B7-1plus B7-2 expressing CHO cells, or control CHO-neo cells only, or withanti-CD28 monoclonal antibody (1 μg/ml), and the time course of cellularproliferation determined. The results are shown graphically in FIG. 20.Proliferation was less vigorous in the presence of 10 nM PMA than whencells were cultured with anti-CD3 mAb (FIG. 20). The time course ofproliferation was similar in cultures that contained B7-1, B7-2 or amixture of cells expressing B7-1 plus B7-2. In contrast, cells culturedin PMA only or PMA plus CHO-neo cells incorporated low amounts ofthymidine, consistent with the rigorous removal of antigen presentingcells. Thus, B7-1 or B7-2 molecules expressed on CHO cells can provide acostimulatory signal to T cells activated either by anti-CD3 mAb or PMA,but proliferation is stronger with anti-CD3 mAb.

Resting T cells do not express detectable amounts of CTLA4, howeveractivated T cells express both CD28 and CTLA4 by day 2 of culture(Linsey et al.(1992) J. Exp. Med. 176:1595). Given that both B7-1 andB7-2 bind to CD28 and CTLA-4, it was possible that some component ofcellular activation could be attributed to CTLA-4, compatible withprevious studies indicating that CTLA-4 ligation can enhance comitogeniceffects of suboptimal CD28 ligation (Damle et al., (1994) J. Immunol.152:2686). For this experiment, anti-CD28 Fab fragments were produced bypapain digestion of 9.3 mAb and two cycles of purification on a proteinA column (Pierce, Rockford, Ill.). CD28⁺ T cells were cultured in thepresence of anti-CD3 coated beads and CHO (as described above) cells forthree days in the presence of increasing amounts of CD28 Fab fragments.Cells were pulsed with tritiated thymidine and scintillation countingperformed on the indicated day of culture. IL-2 concentration in culturesupernatants was determined by ELISA using a commercially available kitfrom T Cell Diagnostics (Cambridge, Mass.) after 24 hours of culture.The values reported were assessed by using dilutions of culturesupernatant that yielded read-outs within the linear portion of thestandard curve.

The results are shown graphically in FIG. 21. Proliferation induced byboth B7-1 and B7-2 was inhibited >95% in a dose-dependent manner by CD28Fab fragments, indicating that both forms of B7 costimulation arecritically dependent on interaction with CD28. IL-2 accumulation inculture supernatants was also efficiently blocked by the Fab fragments.Together these results demonstrate that both B7-1 and B7-2 moleculesexpressed on CHO cells are capable of costimulating T cell proliferationsimilarly to costimulating T cell proliferation with CD28 mAbs.

Example 12

Costimulation with B7-1 and B7-2 Induce Long Term Proliferation of CD4⁺T Cells

The ability of the B7 ligands to sustain T cell proliferation in longterm cultures was investigated. For these experiments, CD4⁺ T cells wereobtained from CD28⁺ T cells by negative selection using magnetic beads(Dynal) coated with CD8 monoclonal antibodies as described in June etal. (1989) J. Immunol. 143:153. The phenotype of the cells was 99%CD2⁺98% CD28⁺, and 96% CD4⁺. 5×10⁶ purified CD4⁺ T cells were stimulatedwith anti-CD3 monoclonal antibody coated beads (1.5×10⁷ beads) andmitomycin C-inactivated CHO cells (2×10⁶ cells) expressing B7-1, B7-2,or neomycin resistance only, or with anti-CD3 plus anti-CD28 coated“cis” beads, i.e. with both antibodies on the same bead. Beads werecoated with anti-CD3 (OKT3) and anti-CD28 9.3 monoclonal antibody witheach antibody added at 150 femtograms per bead. It is important to notethat no cytokines were added to the culture medium so that cell growthwas dependent on secretion of cytokines and lymphokines. Fresh mediumwas added at two to three days intervals with fresh medium to maintaincell concentrations between 0.5-1.5.×.10⁶ T cells/ml; antibody-coatedbeads and CHO cells were not cleared from culture, but were dilutedprogressively until restimulation. The cell cultures were monitored byelectronic cell sizing using a COULTER COUNTER®ZM and Channelyzer model256 (Coulter, Hialeah, Fla.), and restimulated at approximately 7 to 10day intervals (i.e. when the volume of the T cell blasts decreased to<400 fl) with additional beads and mitomycin C-treated CHO cells. ViableT cells were counted and the total number of cells that would beexpected to accumulate displayed, taking into account discarded cells.

The results are shown graphically in FIG. 22. Both B7-1 and B7-2resulted in exponential T cell expansion, and during the first threeweeks of culture both B7 receptors could consistently induce a >3 log ₁₀of expansion that was polyclonal. Together, the above results indicatethat either B7-l or B7-2 can costimulate long-term proliferation in aCD28-dependent manner, independent of a requirement for simultaneousB7-1 plus B7-2 receptor coexpression. To date, no phenotypic differenceshave been detected in the cells that arise from B7-1 or B7-2 stimulatedCD4⁺ T cell expansion. T cells cultured with anti-CD3 and control CHOcells did not undergo sustained proliferation, and the culture wasterminated due to poor viability. Thus, B7-1 and B7-2 expressed on CHOcells induced long term T cell proliferation of activated CD4⁺ T cellssimilarly to costimulation with anti-CD28 mAbs.

Example 13

Differences in Cytokines Secreted from B7 Versus Anti-CD28 Stimulated TCells in Short Term T Cell Cultures

The experiments described in Examples 11 and 12 did not reveal anynotable differences between B7-1, B7-2, or anti-CD28 mAb in the abilityto provide a costimulatory signal for the induction or maintenance of Tcell proliferation. To further test whether anti-CD28 or B7-1 and B7-2mediated distinct costimulatory effects, the accumulation of variouscytokines was examined in T cell subsets. To first assess global effectsof B7 costimulation on T cells, CD28⁺ T cells were stimulated withplastic immobilized anti-CD3 mAb and titered amounts of B7-1 or B7-2 CHOtransfectants or anti-CD28 mAb 9.3 at 1 μg/ml. Anti-CD3 mAb (OKT3) wasprecoated on the culture wells by overnight incubation with a 10 μg/mlsolution. Supernatants of the T cell cultures were collected after 24 hof culture and analyzed by ELISA using commercially available kits. Thekits were obtained from the following sources: IL-2, T Cell Diagnostics,Cambridge, Mass.; TNF-alpha, GM-CSF, IL-4, and IL-5, R&D Systems,Minneapolis, Minn.; Interferon-gamma, Endogen, Boston, Mass. All valueswere assessed by using dilutions of culture supernatant that yieldedread-outs within the linear portion of the standard curve. The cytokineproduction data is summarized below in Table 3.

No cytokines were detected in supernatants from cells cultured in mediaalone, as would be expected with resting T cells. Similarly, anti-CD3did not elicit detectable amounts of IL-2, although low levels of IFNγ,TNF-α and GM-CSF were present after anti-CD3 stimulation alone. Additionof either B7-1 or B7-2 CHO cells resulted in a dose dependent increasein cytokines. The costimulatory effect was most marked in the case ofIL-2, as both B7-1 and B7-2 resulted in 100-fold or more augmentation ofIL-2 secretion in comparison to CD3 plus control CHO cell cultures.There was also a dose-dependent increase in IFN-γ, IL4, TNF-α and GM-CSFsecretion induced by B7-1 and B7-2. Importantly, B7-1 and B7-2costimulation elicited nearly equivalent amounts of all testedcytokines. There were however some differences between the amount ofcytokines secreted by cells stimulated with anti-CD28 mAb as compared toB7-1 or B7-2; most notably, both B7-1 and B7-2 were associated withhigher levels of IL-4 secretion (B7-1, 200; B7-2, 250; anti CD28, <20pg/ml).

TABLE 3 Effects of B7-1 and B7-2 on Lymphokine Production by CD28⁺ TCells. Culture Condition (CHO cell/T cell ratio) IL-2 IFN-γ IL-4 TNFαGM-CSF Medium <60 <10 <20 <30 <100 αCD3⁺CHO-B7-1(0.1) 1790 2640 62 12502490 αCD3⁺CHO-B7-1(0.2) 2250 3880 110 1690 3110 αCD3⁺CHO-B7-1(0.4) 40404430 149 1910 3260 αCD3⁺CHO-B7-1(0.8) 5500 5930 200 2110 3670αCD3⁺CHO-B7-2(0.1) 1620 3470 70 1310 2990 αCD3⁺CHO-B7-2(0.2) 3640 5490145 2040 4040 αCD3⁺CHO-B7-2(0.4) 5826 7640 220 2440 4420αCD3⁺CHO-B7-2(0.8) 6830 8610 250 2410 4260 αCD3⁺CHO-neo(0.1) <60 480 <20670 520 αCD3⁺CHO-neo(0.2) <60 650 <20 690 570 αCD3⁺CHO-neo(0.4) <60 690<20 745 800 αCD3⁺CHO-neo(0.8) <60 690 <20 605 725 aCD3 only <60 280 <20380 280 αCD3⁺CD28 Mab 9.3 1630 860 <20 1760 2090

As shown in Table 3, B7-1 and B7-2-induced cytokine accumulationappeared to plateau at similar ratios of CHO cells to T cells (about 0.4CHO cells: T cell), suggesting that a failure to detect differencesbetween B7 receptors was not due to a differential dose response betweenB7-1 and B7-2. Further, this indicates that neither B7 receptor had beentested under limiting conditions. However, it was possible thatdifferential effects of B7-1 and B7-2 exist, and that this would beapparent only in T cell subsets. Alternatively, it was possible thatintrinsic differences between B7-1 or B7-2 would only be revealed afterrepeated T cell costimulation, to permit possible cellulardifferentiation.

To assess whether distinct T cell subsets might differentially respondto B7-1 or B7-2, CD28⁺ T cells were divided into CD28⁺CD4⁺ and CD28⁺CD8⁺cells and into CD4⁺CD45RO⁺ and CD4⁺CD45RA⁺ subsets. To obtain CD28⁺CD4⁺or CD28⁺CD8⁺ T cells, the CD28⁺ T cells were subjected to a second roundof negative selection using magnetic beads (Dynal) coated with CD8 orCD4 mAb as described in June et al. (1989) J. Immunol. 143:153.CD4⁺CD45RO⁺ and CD4⁺CD45RA³⁰ subpopulations were isolated by subjectingCD28⁺ T cells to negative selection using magnetic beads and anti-CD45ROmonoclonal antibody UCHL1 or anti-CD45RA monoclonal antibody ALB11(Immuntech).

To examine cytokine production, cells were stimulated withplastic-immobilized anti-CD3 Abs and CHO-B7-1 or CHO-B7-2 cells or withplastic beads expressing both anti-CD3 and anti-CD28 (as describedabove). The results are summarized below in Table 4. Table 4 shows thatB7-1 and B7-2 elicited similar amounts of IL-2 secretion from CD4⁺ Tcells. Both receptors stimulated CD8⁺ T cells with equal efficiency,however about 4-fold less IL-2 accumulated in the supernatants from CD8⁺T cells. Anti-CD28 caused potent costimulation of IFNγ in both CD4⁺ andCD8⁺ T cell subsets. B7 receptors could also elicit high levels of IFN-γfrom either subset, although the magnitude of the effect was two tofour-fold less than anti-CD28 mAb stimulation.

TABLE 4 Effects of B7-1 and B7-2 on lymphokine production by CD4⁺andCD8⁺ T cells (cytokine concentrations in pg/ml) Culture Condition IL-2IFN-γ GM-CSF medium <60 <15 <15 CD4⁺ Cells aCD3 93 1270 9770 αCD3⁺CD28mAbs 87500 44200 77300 αCD3⁺CHO-neo 60 1760 9380 αCD3⁺CHO-B7-1 532014200 32400 αCD3⁺CHO-B7-2 3940 18300 30100 CD8⁺ Cells aCD3 133 1620 8900αCD3⁺CD28 mAbs 53600 26200 69600 αCD3⁺CHO-neo 100 3530 8830αCD3⁺CHO-B7-1 1360 10700 20600 αCD3⁺CHO-B7-2 1320 14600 20700

It was surprising that CD8⁺CD28⁺ T cell cultures accumulated similaramounts of IL-2, IFN-γ and GM-CSF as did CD4⁺CD28⁺ cells. The CD8⁺subpopulation contained <2% CD4⁺ cells, and thus contamination of theCD8⁺ cells by CD4⁺ cells can not explain the nearly equivalent levels ofcytokine secreted by the CD4 and CD8 subsets. In the experiment shown inTable 4, cells were stimulated with beads expressing both anti-CD3 andanti-CD28 mAbs (“cis” stimulation), while in the experiment shown inTable 3, plastic-immobilized anti-CD3 plus fluid phase anti-CD28(“trans”) costimulation was used. “Cis” stimulation was consistentlymore efficient at eliciting cytokine accumulation (Table 4 vs. Table 3,and see FIGS. 25 and 26 below).

The costimulatory signals provided by anti-CD28, B7-1 and B7-2 weresimilarly potent in eliciting accumulation of GM-CSF (Tables 3 and 4).This is most apparent if the fold elevation above anti-CD3 mAb only, orcontrol CHO cultures is examined when comparing cultures stimulated withanti-CD28 or B7-1 and B7-2. With regard to TNFα, both B7-1 and B7-2 hadsimilar costimulatory effects on CD4⁺ T cells.

While the effects of B7-1 and B7-2 appeared quite similar on CD4⁺ andCD8⁺ T cell subsets, it remained possible that distinct functions mightbe revealed in CD4 subsets. Purified CD4⁺ T cells were magneticallysorted into “virgin” CD45RA⁺ and “memory” CD45RO⁺ populations asdescribed above. The cells were then tested for their ability to secreteIL-2, IL-4 and IFNγ after stimulation with anti-CD3 mAb coated beads inthe presence of B7-1 or B7-2-expressing CHO or control neo-CHO cells(see FIGS. 23 and 24). The experiment in FIG. 23 was carried out in R10medium and the experiment of FIG. 24 was carried out in Aim V medium.Supernatants were harvested after 24 hours of culture, and cytokineconcentrations were measured by ELISA as described above. Both B7-1 andB7-2 were able to costimulate both subsets to equivalent levels ofproliferation. However, striking differences were uncovered in theinduction of cytokine secretion by B7 from CD45RO⁺ and CD45RA⁺subpopulations. B7-1 and B7-2 costimulation resulted in the secretion oflarge amounts of IL-2 from both subsets (FIG. 23, left hand panels). Incontrast, neither B7-1 nor B7-2 could elicit IL-4 (FIGS. 23 and 24,right hand panels) or IFNγ (FIG. 24, left hand panels) from the CD45RA⁺subpopulation. Since both B7-1 and B7-2 were tested at a variety of Tcell to CHO cell ratios, ensuring that responses were assessed atplateau levels of costimulation, it is unlikely that either receptor hasa differential costimulatory function on CD4⁺ T cells with naive andmemory phenotypes. Furthermore, no differential sensitivity or primingof these cellular subsets to low levels of B7 stimulation was observed;B7-1 and B7-2 mediated lymphokine responses occurred at similarthresholds in both naive and memory subsets.

Example 14

Differential Cytokine Secretion upon Costimulation with Anti-CD28 in“cis”, in “trans”, or with B7 Molecules in Long Term Culture

The experiments described in Examples 11, 12 and 13 indicate that B7receptors have similar costimulatory effects on the cytokines producedduring the first round of T cell activation and division, and indicatethat CD4 subpopulations have differential capacity to secrete cytokines.In this example, differences in the ability of B7-1 or B7-2 to inducedifferentiation were investigated. CD4⁺CD28⁺ T cells were stimulatedwith anti-CD3 in the presence of CHO cells expressing B7-1 or B7-2, orwith anti-CD3 plus CD28 mAbs on beads in “cis” as described in Example12. The cells were maintained in exponential growth during theexperiment. Supernatants were collected after 24 hours of culture and onday 11, cells were washed and placed in fresh medium, restimulated withfresh anti-CD3 and CHO cells, and supernatants collected after a further24 hours culture. Cytokine levels were measured as described above.

The results are shown graphically in FIGS. 25-27. During the initial 24hours of activation, B7-1 and B7-2 induced nearly equivalent amounts ofIL4, and this was more than that induced by anti-CD28 (FIG. 25, bottompanels). In contrast, costimulation with B7-1 and B7-2 did not elicit asmuch IL-2 as anti-CD28 stimulation during the first 24 hours (FIG. 25,top). These results are consistent with Table 4, where anti-CD28stimulation in “cis” was also employed. However, on restimulation ofcells with B7 receptors on day 12 of culture, about 10-fold more IL-4accumulated when compared to the initial stimulation of resting T cells.The anti-CD28 mAb mediated increase in IL-4 secretion on restimulationwas not as striking as with B7 restimulation. In contrast, anti-CD28 wasmore efficient than B7 in the induction of IL-2 secretion duringrestimulation.

IL-5 secretion was not detectable after primary stimulation withanti-CD3 and anti-CD28, while both B7-1 and B7-2 resulted in low-levelIL-5 secretion of similar magnitude during the first day of stimulation(FIG. 25, bottom). A notable increase in B7-1 and B7-2-mediated IL-5secretion occurred on day 12 restimulation. IL-5 secretion alsoincreased after anti-CD28 restimulation, however, the increase was about8 to 10-fold less than that due to B7 restimulation. Thus, the patternof IL-5 secretion is similar to that of IL-4 (FIG. 25 vs. 26, bottompanels).

The effects of restimulation on IFNγ were also examined. High level IFNγsecretion occurred within 24 hours after B7 and anti-CD28 primarystimulation (FIG. 26, top), consistent with Table 4. At restimulation,however, anti-CD28 was superior to both B7 receptors. Thus, the patternof anti-CD28 and B7-mediated IFNγ secretion is similar to that of IL-2,and the pattern of IL-5 secretion is similar to that of IL-4 (FIGS. 25vs. 26).

FIG. 27 shows the effects of anti-CD28 and B7 restimulation on GM-CSFand TNFα secretion by CD4⁺ T cells. With regards to GM-CSF, bothanti-CD28 and B7-1 and B7-2 increased GM-CSF secretion, although thefold costimulation was more modest, at 4 to 8-fold over that induced byanti-CD3 plus control CHO cells (FIG. 27, bottom). On restimulation,there were no consistent differences between the various CD28 ligands inthe ability to promote GM-CSF secretion. In contrast, anti-CD28 was moreeffective than B7 receptors at maintaining TNFα secretion onrestimulation (FIG. 27, top). Thus, during the initial activation of Tcells, and during reactivation of CD4⁺ T cell blasts in vitro, noconsistent differences between B7-1 and B7-2 could be identified in anyof the cytokines examined. Interestingly however, anti-CD28 mAb favoredIL-2, IFNγ, and TNFα secretion, while B7-1 and B7-2 promoted IL-4 andIL-5 secretion. Thus, costimulation with B7-1 or B7-2 resulted inpreferential secretion of TH2-specific cytokines, whereas costimulationwith anti-CD28 resulted in preferential secretion of TH1-specificcytokines.

The above results did not reveal any consistent differences in theinduction of cytokine secretion by B7-1 and B7-2 while differencesbetween anti-CD28 and B7 were observed in FIGS. 25-26 and in Table 3. Todetermine a potential mechanism for these differences, CD28⁺ T cellswere cultured in the presence of anti-CD3 plus anti-CD28 beads in “cis”(both antibodies on the same bead) or in “trans” (both antibodies ondifferent beads). For “cis” stimulation, immunomagnetic beads werecoated with OKT3 and 9.3 mAbs with each antibody added at 150 femtogramsper bead and added at a ratio of 3 beads per T cell. For “trans”stimulation, an equal amount anti-CD3 and anti-CD28 coated beads wereadded at a ratio of 3 (of each type) beads per T cell. In addition,cells were cultured with anti-CD3 plus anti-CD28 beads in “cis” withmitomycin-inactivated CHO-neo cells added at a ratio of 2.5:1 T cell toCHO cell, as a control for factors intrinsic to CHO cells. Finally, Tcells were cultured with anti-CD3 beads and CHO-B7-1 cells at a ratio of2.5:1 T cell to CHO cell. The cells were cultured as indicated inExample 12.

The results of these experiments are shown graphically in FIGS. 28-29.All conditions induced exponential expansion of CD4 T cells (FIG. 29).However, there were differences in the cytokines induced by these formsof costimulation (FIG. 28). Cells stimulated with anti-CD28 in “cis” orwith anti-CD28 in “cis” plus CHO-neo cells maintained high levels ofIL-2 secretion. In contrast, anti-CD28 stimulation in “trans” was lessefficient at inducing IL-2 secretion, and this form of costimulation wasprogressively less efficient upon repetitive restimulation. The anti-CD3plus CHO-B7-1 stimulation in “trans” was the only condition thatresulted in progressively increasing amounts of IL-4 secretion,consistent with the results shown in FIG. 26 and Table 3. Together, theabove results demonstrate that B7-1 and B7-2 both have the ability tostimulate T cell proliferation and cytokine secretion and that themanner of CD28 costimulation can have substantial effects on thepatterns of cytokine secretion.

Example 15

Cell Death by Apoptosis of CD8⁺ T Cells Costimulated with Anti-CD28

Previous information in this application shown in FIG. 4 has shown thatCD28 costimulation can favor the growth of CD4⁺ T cells. To determine amechanism for this effect, CD8⁺ T cells were isolated by negativeimmunomagnetic selection. The cells were stimulated with anti-CD3antibody coupled to beads (b) or to a solid phase (SP) i.e., tissueculture dish plus anti-CD28 in “cis” or in “trans”. The induction ofapoptosis was assessed by detecting single strand DNA breaks using aflow cytometric TdT assay (Gorczyca, W., J. Gong, and Z. Darzynkiewicz1993. Detection of DNA strand breaks in individual apoptotic cells bythe in situ terminal deoxynucleotidyl transferase and nick translationassays. Cancer Res. 53:1945). During the first 48 hours of culture (S1),the cells became activated and <10% of cells underwent apoptosis (Table5). The cells were restimulated twice with anti-CD3 and anti-CD28 whenrequired and the induction of apoptosis again assesed 24 to 48 hoursfollowing each restimulation (S2 and S3). A dramatic increase in DNAbreaks was uncovered. This was not prevented with the addition ofrecombinant IL-2 (rIL-2) at 100 U/ml.

TABLE 5 Apoptosis assays of CD8⁺ T cells costimulated with anti-CD28 %TdT Positive by Flow Cytometry Stimulation T0 T24 hr T48 hr S1 OKT3/9.33b/c 2 6 1 OKT3 3b/c + 9.3 3b/c 2 3 1 OKT3 3b/c + soluble 9.3 2 3 1OKT3sp + soluble 9.3 2 1 2 Medium 2 2 2 S2 OKT3/9.3 3b/c + rhIL2 40 1539 OKT3 3b/c + 9.3 3b/c 22 47 63 OKT3 3b/c 9.3 3 3b/c + rhIL2 22 55 34OKT3 3b/c + soluble 9.3 27 70 72 OKT3 3b/c + soluble 9.3 + 27 76 59rhIL2 S3 OKT3/9.3 3b/c 67 OKT3/9.3 3b/c + rhIL2 11 75 OKT3 3b/c + 9.33b/c + rhIL2 19 52

It was next determined if the induction of apoptosis in CD8⁺ T cells wasspecific to the CD8⁺ T cell subset. CD4⁺ and CD8⁺ T cells were isolatedand stimulated separately with anti-CD3 and anti-CD28 antibodies. Duringthe first 48 hours of culture (S1), there was little evidence ofprogrammed cell death in either the CD4 or the CD8⁺ T cell subsets. Incontrast, on restimulation (S2), there was a marked induction of celldeath in the CD8⁺ T cell subset (Table 6). Again, this was not preventedby addition of exogenous IL-2. Thus, the selective induction of CD8⁺ Tcell death is one mechanism that permits CD28 stimulation to enhanceCD4⁺ T cell expansion. The absence of programmed cell death in the CD4⁺T cells is consistent with the observations shown elsewhere in thisapplication that the CD4⁺ cells remain polyclonal for extended periodsof culture.

TABLE 6 Apoptosis assays of CD4 and CD8⁺ T cells % TdT Positive by FlowCytometry CD4 and CD8 T cells T0 T24 hr T48 hr S1 CD4 OKT3/9.3 3b/c(cis) 5 10 9 CD4 OKT3 3b/c + rhIL2 5 9 9 CD4 OKT3 3b/c + 9.3 3b/c(trans) 5 12 11 CD8 OKT3/9.3 3b/c (cis) 3 6 3 CD8 OKT3 3b/c + rhIL2 3 612 CD8 OKT3 3b/c + 9.3 3b/c (trans) 3 7 9 S2 CD4 OKT3/9.3 3b/c (cis) 618 13 CD4 OKT3 3b/c + rhIL2 4 27 9 CD4 OKT3 3b/c + 9.3 3b/c (trans) 6 1315 CD8 OKT3/9.3 3b/c (cis) 8 63 47 CD8 OKT3 3b/c + rhIL2 15 75 62 CD8OKT3 3b/c + 9.3 3b/c (trans) 20 79 61

The selective cell death of CD8⁺ T cells and not CD4⁺ T cells will beuseful in selectively enriching a population of T cells, for exampleCD28⁺ T cells in CD4⁺ T cells during expansion.

Example 16

Expansion of CD4⁺ T Cells from HIV Infected Individuals

In order to determine whether CD4⁺ T cells from individuals infectedwith HIV can similarly be expanded ex vivo, CD4⁺ T cells were obtainedfrom HIV infected individuals and activated with anti-CD3 and anti-CD28coated beads (3 beads containing an equimolar amount of each antibody,per T cell) in the presence or absence of anti-retroviral drugs. CD4⁺ Tcells from a patient with 430 CD4⁺ T cells/μl were isolated by negativeselection as described in Example 12 and incubated in standard RPMI 10%fetal calf serum either in the presence or absence of the followinganti-retroviral drugs: AZT (available from Burroughs-Wellcome) at 1 μM,DDI (available from Bristol Myers Squibb) at 10 μM, and Nevirapine(available from Boehringer Mannheim) at 1 μM. The growth curves of thecells are represented in FIG. 30. The T cells expanded exponentially bya factor of more than 10,000 fold either in the presence or absence ofadded anti-retroviral drugs. Moreover, the CD4⁺ T cells expanded tohigher numbers in the absence of the drug. In addition, no significantamounts of HIV-1 was detected in either culture, as the amount HIVp24present in the supernatant of the cultures (determined by theSpearmen-Karber method) was <50 pg/ml. The Spearmen-Karber method isdescribed in Richman D. B., Johnson V. A., Mayrs V. L. 1993, In vitroevaluation of experimental agents for anti-HIV activity. CurrentProtocols in Immunology ch. 12.9, Colligan J. E., Kruisback A. M.,Margolis D. H., Shevach E. M., Strober W. Ed. Greene and Wiley.Interscience NY.

To further investigate the finding that the amount of HIV produced inthe CD4⁺ T cell cultures stimulated in the presence of anti-CD3 andanti-CD28 was very low, the extent of replication of the virus wascompared directly between CD4⁺ T cells isolated from HIV (US 1 isolate)infected cells (according to Richman D. B., Johnson V. A., Mayrs V. L.1993, In vitro evaluation of experimental agents for anti-HIV activity.Current Protocols in Immunology ch. 12.9, Colligan J. E., Kruisback A.M., Margolis D. H., Shevach E. M., Strober W. Ed. Greene and Wilcy.Interscience NY), stimulated with PHA (5 μg/ml) and IL-2 (100 U/ml) orwith anti-CD3 and anti-CD28. The TCID₅₀ was determined by the method ofSpearmen-Karber at days 7, 14, and 21 of the cultures. As shown in Table7, there was a marked difference between the titer of virus in thesupernatants of T cells stimulated with anti-CD3 and anti-CD28 coatedbeads (3 beads per T cell) as compared to the conventional method ofpropagation using PHA and IL-2. The mechanism for this interestingeffect is not yet known, but it suggests another potential mechanism for“rescuing” uninfected CD4 cells in cultures from HIV seropositivepatients.

TABLE 7 CD4⁺ T Cells stimulated by anti-CD3 and anti-CD28 do not supportreplication of HIV-1. TCID₅₀ Cell Stimulation Day 7 Day 14 Day 21anti-CD3 + anti-CD28 <64 <64 <64 PHA + IL-2 18820 >65000 >65000

It was possible that CD28 causes the proliferation of a particularsubset of lymphocytes that are resistant to infection by HIV-1.Alternatively, it was possible that the mechanism of stimulation per sewas able to confer resistance or sensitivity to HIVinfection/expression. To test these possibilities, PBMC were obtainedfrom a normal blood donor, and either the purified CD4⁺ T (10⁵cells/well) cells or whole PBMC (10⁵ cells/well) activated with PHA (5μg/ml) or with anti-CD3 and anti-CD28 coated beads (3 beads per T cell).The cells were infected with a T cell trophic variant of HIV-1(US1) or amonocyte trophic variant (BAL) on day 2 of culture as described inRichman D. B., Johnson V. A., Mayrs V. L. 1993, In vitro evaluation ofexperimental agents for anti-HIV activity. Current Protocols inImmunology ch. 12.9, Colligan J. E., Kruisback A. M., Margolis D. H.,Shevach E. M., Strober W. Ed. Greene and Wiley. Interscience NY. Thelevel of virus expression was quantitated in the culture supernatants onday 7 as shown in Table 8 by the Spearmen-Karber method. In PBMC, highlevels of virus were expressed if the cells were stimulated with PHAwhereas very low or no levels of virus were detected in culturesstimulated with anti-CD3 and anti-CD28 antibodies. This result wasobtained whether or not plastic adherent monocytes/macrophages (M/M)were added to the culture (10⁴ cells per well).

TABLE 8 Virus titrations in PHA and CD3/CD28-stimulated PBMCs with orwithout addition of monocytes/macrophages. US1 TCID50 BAL TCID50 CellGroup Day 7 Day 7 PBMC + PHA 332555 241029 PBMC + anti-CD3/CD28 279 77CD4 cells + M/M + PHA >390625 >390625 CD4 cells + M/M + anti- 386 279CD3/CD28 CD4 cells anti- 1012 386 CD3/CD28

Thus, stimulation of CD4⁺ T cells infected with HIV with anti-CD3 andanti-CD28 results in much lower amounts of HIV particles produced ascompared to the conventional method of T cell stimulation with PHA andIL-2. Moreover, since exponential growth of the cells was observed forat least 40 days, this method could therapeutically be useful for exvivo and in vivo expansion of CD4⁺ T cells from an individual infectedwith HIV.

Example 17

Large Scale Expansion of CD4⁺ T Cells Using Anti-CD3 and Anti-CD28Antibodies

To determine if the small scale expansion of T cells with anti-CD3 andanti-CD28 antibodies is also functional on a larger scale, required forclinical use, CD4⁺ T cells were obtained from a normal donor andcultured in either 3 liter gas-permeable bags (Baxter) or in T75 flasks(FIG. 31). The large scale culture system was seeded with 5×10⁷ cells.The cells were stimulated with a bead:cell ratio of 3:1 and the beadscontained an equimolar amount of anti-CD3 (OKT3) and anti-CD28 (mAb 9.3)antibodies. The cells cultured in T75 were restimulated at day 12 (S2)wit anti-CD3 and anti-CD28 coated beads in the same conditions as forthe first stimulation. The cells cultured in the gas-permeable bags wererestimulated at day 11 (S2). No exogenous cytokines were added to thelarge scale culture system. The T75 flasks were carried out induplicate, one containing exogenous IL-2 (100 μ/ml) and the other withno added cytokine. The large scale culture grew equivalent to the smallscale culture system, and was harvested on day 19 of culture and2.4×10¹⁰ CD4⁺ T cells recovered. Viability was >95%. Thus, CD4⁺ T cellscan be expanded to high cell numbers in large cultures by stimulatingthe T cells with anti-CD3 and anti-CD28 coated beads, and will thus beuseful for clinical uses.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method for including ex vivo proliferation of a population of Tcells to sufficient numbers for use in therapy, comprising contacting apopulation of T cells ex vivo with a surface having directly attachedthereon: (a) an anti-CD3 antibody or CD3-binding fragment thereof, whichprovides a primary activation signal to the T cells, thereby activatingthe T cells; and (b) an anti-CD28 antibody or CD28-binding fragmentthereof, which stimulates a CD28 accessory molecule on the surface ofthe T cells, thereby stimulating the activated T cells, wherein theanti-CD3 antibody or CD3-binding fragment thereof, and the anti-CD28antibody or CD28-binding fragment thereof, are directly attached on thesame surface, the anti-CD3 antibody or CD3-binding fragment thereof, andthe anti-CD28 antibody or CD28-binding fragment thereof, therebyinducing the T cells to proliferate to sufficient numbers for use intherapy.
 2. The method of claim 1, wherein the anti-CD3 antibody orCD3-binding fragment thereof is an anti-human CD3 monoclonal antibody orCD3-binding fragment thereof.
 3. The method of claim 1, wherein theanti-CD28 antibody or CD28-binding fragment thereof is an anti-humanCD28 monoclonal antibody or CD28-binding fragment thereof.
 4. The methodof claim 1, further comprising: monitoring the proliferation of the Tcells; and reactivating and re-stimulating the T cells with the anti-CD3antibody or CD3-binding fragment thereof, and the anti-CD28 antibody orCD28-binding fragment thereof, when the rate of T cell proliferation hasdecreased to induce further proliferation of the T cells.
 5. The methodof claim 4, wherein the step of monitoring proliferation of the T cellsis by examining cell size or determining the level of expression of acell surface molecule selected from the group consisting of B7-1, B7-2,and any combination thereof, and the step of reactivating andrestimulating is initiated when T cell has decreased or when the levelof the cell surface molecule has decreased.
 6. The method of claim 1,wherein the T cells are induced to proliferate to about 100-fold theoriginal T cell population.
 7. The method of claim 1, wherein the Tcells are induced to proliferate to about 100,000-fold the original Tcell population.
 8. The method of claim 1, wherein the T cells areinduced to proliferate for at least 3 days.
 9. The method of claim 1,wherein the T cells are induced to proliferate for at least 7 days. 10.The method of claim 1, wherein the surface is a bead.
 11. The method ofclaim 10, wherein the bead is a magnetic bead.
 12. The method of claim10, wherein the bead is a polystyrene bead.
 13. The method of claim 1,wherein the surface is a cell surface.
 14. The method of claim 1,wherein the surface is a tissue culture dish.
 15. The method of claim 1,wherein the population of T cells are induced to proliferate tosufficient numbers for use in treating cancer.
 16. The method of claim1, wherein the population of T cells are induced to proliferate tosufficient numbers for use in treating an infectious disease.
 17. Theanti-CD28 antibody of claim 3, wherein the antibody is 9.3, produced bythe hybridoma assigned ATCC No. HB-10271.
 18. The anti-CD28 antibody ofclaim 3, wherein the antibody is EX 5.3D10, produced by the hybridomaassigned ATCC No. HB-11373.